Biofabrication最新文献

Cartilage tissue engineering requires biomaterials that can effectively maintain the tissue-specific functions of chondrocytes to enable the restoration of cartilage structure and function. Decellularised extracellular matrix (dECM)-derived hydrogels serve as tissue-specific biomaterials capable of preserving native biochemical cues and maintaining physiological chondrocyte phenotype in three-dimensional culture. However, their sol-gel transition relies heavily on collagen fibrillogenesis, a slow and poorly controllable process that limits mechanical tunability and suffers from inter-batch variability. Therefore, further efforts are required to functionalise cartilage dECM to achieve reproducible and controllable physicochemical properties. Here, we present a light-activated cartilage dECM hydrogel system based on ruthenium/sodium persulfate (Ru/SPS)-mediated dityrosine crosslinking, enabling rapid hydrogel formation under visible light irradiation while providing tunable mechanical properties and improved biological functionality. Comparison of the decellularisation protocols indicated that Triton X-100 combined with ammonium hydroxide efficiently eliminated residual DNA while preserving a substantial proportion of the native cartilage proteome. Pepsin-solubilised cartilage dECM hydrogels formed via dityrosine-based photo-crosslinking exhibited rapid gelation behaviour and superior mechanical characteristics compared to conventional thermally gelled dECM. The photo-crosslinked dECM hydrogels were cytocompatible, supported human bone marrow-derived mesenchymal stem cells (hBMSCs), and favoured cartilage-specific phenotypes, as demonstrated by the upregulation of chondrogenic genes, includingCOL2A1andACAN, compared with gelatin methacrylate (GelMA) hydrogels. Importantly, this photo-crosslinking strategy overcomes the incompatibility between oxygen-sensitive redox-based photochemistry and hypoxic culture conditions, enabling the incorporation of oxygen-scavenging microcapsules to establish low-oxygen microenvironments. Under hypoxia, the cartilage dECM hydrogels promoted a more articular-like phenotype in hBMSC-derived chondrocytes, with transcriptomic features associated with TGF-β/SMAD2/3 and IGF-1/2-IGF-1R signalling. Collectively, these findings establish photo-crosslinked cartilage dECM hydrogels as a biomaterial platform with tunable mechanical properties and favourable biological functionality for cartilage tissue bioengineering and biomimeticin vitrocartilage models.

Three-dimensional (3D) cell spheroids are widely used asin vitrotissue models, yet quantitative understanding of their morphogenesis remains limited. We present an integrated experimental-computational framework to analyze, model, and modulate the compaction of cell aggregates in agarose microwells of defined geometries. Custom 3D-printed stamps produced circular, square, and triangular microwells of equal cross-sectional area. Time-lapse imaging combined with AI-based segmentation enabled tracking of spheroid morphology, with circularity and projected area serving as quantitative descriptors of compaction. The process followed predictable exponential kinetics, with mesenchymal (HDF) spheroids compacting faster than epithelial (ARPE-19) ones. Computational fluid dynamics (CFD) simulations modeled spheroid rounding as a visco-capillary-driven process, where the extracted visco-capillary velocity unified experimental and simulated dynamics. Mechanical measurements by atomic force microscopy and compression confirmed that differences in surface tension predominantly governed the observed kinetics. Pharmacological modulation of cytoskeletal tension revealed that inhibition of contractility markedly altered spheroid formation dynamics, enabling the generation of stable, non-spherical aggregates. Using this principle as a shape-engineering strategy, we produced aggregates with distinct geometries (brick-like, prismatic, and star-shaped), characterized by an increased surface-to-volume ratio compared to conventional spheroids. Limitations of the approach include the use of pharmacological cytoskeletal modulation and constraints in geometric fidelity arising from printing resolution, agarose casting, cell filling, and intrinsic smoothing of sharp features during cell aggregation. Collectively, this work establishes a geometry-controlled platform for quantitative analysis of spheroid formation and mechanical behavior, and provides a versatile framework for designing cell aggregates with defined shapes.

Osteochondral defects present substantial clinical challenges due to the complex, multilayered structure and distinct physiological properties of cartilage and subchondral bone. Here, we report a three-dimensional (3D)-printed osteochondral scaffold featuring a dual biomimetic design that integrates vertically oriented microchannels with bioinspired nano-mineral precursors. Specifically, a multifunctional hierarchical construct was developed by incorporating ultrasmall (∼1 nm) polymer-induced liquid precursor-modified amorphous calcium phosphate (nCaP) into a GelMA-based matrix. Using digital light processing-based 3D printing, a biphasic scaffold with spatially defined architectures was fabricated, consisting of a pure GelMA upper layer featuring combined lotus-like and radial pore distributions to emulate the cartilage microenvironment, and a nCaP/GelMA lower layer with lotus-like pore architecture to support subchondral bone regeneration. Notably, in contrast to conventional inorganic fillers such as nanohydroxyapatite (nHAp), the incorporation of ultrasmall nCaP nanoclusters did not adversely affect photopolymerization behavior or printing fidelity, thereby enabling high-resolution fabrication. Beyond structural advantages, nCaP incorporation markedly enhanced the bioactivity of the scaffold. Compared with nHAp, nCaP significantly promoted the recruitment and osteogenic differentiation of endogenous bone marrow-derived mesenchymal stem cells, while also facilitating extracellular matrix deposition, mineralization, and angiogenesis. Transcriptomic analysis further indicated that these effects were associated with the upregulation of angiogenic factor EGFL6, suppression of inflammation-related TNFSF14/NF-κB signaling, and activation of the PI3K-Akt pathway. Collectively, bothin vitroandin vivoevaluations demonstrated that the nCaP/GelMA scaffold achieved improved tissue integration, restoration of hierarchical architecture, and enhanced mechanical performance compared with control groups. These findings underscore the potential of dual biomimetic scaffold design as an effective strategy for osteochondral regeneration.

Pancreatic cancer-associated cachexia is marked by adipose tissue wasting, thermogenic remodeling, and a state of hypermetabolism, yet robust preclinical models to study these phenomena are lacking. In this study, we present a spatially defined three-dimensional (3D) core-shell microcuboid coculture platform designed to investigate the interaction between adipocytes and pancreatic cancers. This innovative system consists of differentiated white adipocytes at the core, surrounded by pancreatic ductal adenocarcinoma (PDAC) cells embedded in 3D-printed microcuboids, arranged concentrically within a collagen coculture matrix construct. Within this framework, we observed significant enhancement of adipocyte lipolysis and browning, as evidenced by BODIPY dye-tracked lipid migration, sustained glycerol release, and progressive expression of extracellular UCP1 or the mitochondrial brown fat uncoupling protein 1, particularly pronounced in cocultures involving aggressive pancreatic cancer cell lines. The integrity of the core-shell architecture persisted for up to 21 d but progressively disintegrated under the influence of the cancer cells marked by cancer cell invasion into the adipocyte regions. Gene profiling revealed a downregulation of adipogenic markers, such asPparg, Plin1, andLipe, alongside an increase inUcp1transcripts, suggesting a metabolic shift from lipid storage to utilization and thermogenic activation. In contrast to existing 3D engineered systems, our platform offers enhanced long-term viability, controlled compartmentalization, mechanical tunability, and high spatiotemporal resolution. It effectively recapitulates the dynamic interplay between cancer and adipose cells, along with the catabolic characteristics of PDAC-associated cachexia, serving as a scalablein vitrotool for mechanistic investigations, and for testing potential anti-cachexia interventions, filling the gap between simplisticin vitroassays and complex animal models.

To replicate the function of native tissue in cell cultures, one must reproduce the structure of the native tissue. This can be achieved using tissue-guiding architectures with cell-scale dimensions, typically ranging from single to tens of microns. However, this spatial resolution exceeds the capabilities of many common fabrication methods, including extrusion-based 3D printing. Indeed, although increasingly popular in bioengineering, extrusion-based 3D printing is not only limited by the properties of the print materials, but also by the inherent trade-off that smaller features require smaller nozzles. This, in turn, results in more toolpaths and longer build times. To overcome this limitation, we introduce nozzles with micro-scale structures at their orifice, fabricated through straightforward hot embossing of commercial polypropylene nozzles. This approach enables microstructure printing using large (⩾0.4 mm inner diameter) nozzles. Specifically, we demonstrate rapid printing of microstructured soft substrates, capable of guiding skeletal and cardiac muscle cell cultures into physiomimetic, anisotropic tissues for electrophysiological assays and drug studies. Furthermore, we show that axonal growth in neuronal tissue cultures can also be directed. Thus, our approach may serve as a scalable and easily accessible method for fabricating human cell cultures and tissue models with enhanced physiological relevance.

Human hepatic organoids derived from pluripotent or adult stem cells offer powerful platforms for disease modeling and drug discovery. However, developing robust and scalable organoids capable of sustaining long-term functionality remains challenging. Here, we developed a novel, semi-defined approach using a self-assembling peptide and collagen I to create highly uniform human induced pluripotent stem cell-derived hepatic organoids in droplet format, which we term hepatic organobodies (OBs). This method enabled rapid, reproducible production of threedimensional (3D) liver tissues, which remained structurally, metabolically, and functionally stable for several weeks. OBs adopted hallmark hepatic morphology and expressed key hepatocyte genes, several at levels approaching freshly isolated primary human hepatocytes (PHHs). OBs secreted substantially higher albumin and A1AT compared with parallel two dimensional cultures, and transcriptomic profiling revealed marked enhancement of hepatic maturation, including elevated expression ofCYP3A4, CYP2C9, andCYP1A2, and enrichment of PPAR signaling and fatty acidβ-oxidation pathways. Additionally, OBs exhibited drug metabolizing activity comparable to classical Matrigel-based organoids and demonstrated CYP3A4 and CYP2C9 activities comparable to the 'gold standard' 3D PHH microtissues. Critically, OBs accurately predicted hepatotoxicity of more than 10 reference compounds, outperforming HepG2 cells and matching PHH-based benchmarks. Overall, we present OBs, a novel, and scalable 3D liver model that delivers advanced maturation and robust metabolic function. This platform offers a powerful and reproducible alternative to existing organoid systems as it avoids animal-derived, undefined matrices such as Matrigel, requires no specialized equipment, and relies on rapid self-curation of the hydrogel triggered by physiological salt concentrations, making the process fast, reproducible, broadly accessible, and scalable.

Bioprinting processes have greatly advanced in recent years through improvements in print accuracy and bioink optimization. Despite these advances, optimizing cell distribution and viability still relies on guess-and-check methods and destructive post-printing testing. The ability to monitor cells during printing would improve print quality and inform complex bioprinting processes, such as the generation of cellular gradients or controlled bioink transitions. Real-time monitoring using dielectric impedance spectroscopy (DIS) alleviates this burden by correlating impedance|Z|to cell properties. However, the influence of bioink properties on these measurements is unknown. Using an in-line impedance sensor, we assessed the effects of alginate bioink concentration, pH, and crosslinking on impedance over 1-25 000 kHz and determined how these properties influenced the detection of primary chondrocytes. In each scenario, impedance was highest in samples with low alginate concentration, low sample pH, or crosslinker. In nearly all samples, the addition of cells resulted in an increase in impedance compared to acellular samples, and this difference in impedance was used to quantify cell presence, termed |Z_cells|. Higher alginate concentrations at 1 w/v% and 3 w/v% showed greater |Z_cells|, indicating reliable cell detection. Although |Z_cells| varied greatly with alginate or phosphate-buffered saline pH, similar measurements were found in pH resembling cell media. Optimal frequency ranges for monitoring acellular and cellular samples were from 10-100 kHz and 1000-25 000 kHz. Furthermore, cells were detected in real-time as acellular and cellular alginate bioinks were transitioned during bioprinting. This transition in cell concentration was spatially mapped to deposited bioink, providing a visual display of bioink transition using impedance. In summary, DIS detected cells suspended in alginate bioink and showed potential for real-time mapping of cell deposition.

Dermo-epidermal skin substitutes (DESS) offer a promising approach for treating full-thickness skin defects, but prolongedin vitroculture leads to significant contraction of the engineered tissue, particularly in the presence of an epidermal layer and highly contractile donor cells. This compromises graft quality and reproducibility, posing a challenge for preclinical research. To overcome this limitation, we developed a customized anti-shrinkage device (ASD) designed to physically constrain the substitute while remaining compatible with the established fabrication process of plastically compressed DESS. Skin substitutes were cultured with and without the ASD, and their contraction behavior, morphology, and cellular organization were analyzed. Our results showed that the ASD effectively minimized tissue shrinkage (3%-8%, depending on the experimental settings), preserving morphology and reducing variability compared to non-constrained substitutes, which exhibited significant contraction (23%-36%) and irregular morphology. Fibroblasts in contraction-protected substitutes maintained an elongated, spindle-shaped morphology without pathological myofibroblast differentiation, as indicated by the absence ofα- smooth muscle actin expression. Furthermore, the epidermal layer in contraction-protected substitutes exhibited improved structural organization. Overall, the ASD provides a user-friendly and effective engineering solution to mitigate contraction in bioengineered skin substitutes, enhancing their stability and reproducibility for preclinical applications. This approach may contribute to improving the reliability of advanced skin grafts for future clinical use.

Hydrogels, possessing biocompatibility and flexibility, are widely used across biomedical and industrial domains, with their concentration serving as a critical determinant of their physicochemical properties. However, conventional methods for concentration assessment exhibit significant limitations; invasive techniques damage the original state of the sample, while existing non-invasive approaches often lack precision at extreme concentration levels. To address these challenges, this study introduces a novel, highly accurate, non-invasive ultrasound-based methodology for hydrogel concentration analysis. A single-element ultrasound transducer was used to collect concentration data while preserving sample integrity. This approach mitigates the accuracy variation observed in existing technologies, enabling precise classification across all concentration levels. In particular, complex ultrasound signal pattern analysis was conducted using a convolutional neural network-based machine learning framework, achieving concentration classification with an accuracy exceeding 99%. Through highly accurate and non-destructive concentration classification, the proposed method holds substantial potential as a core technology for improving the quality control of hydrogel-based constructs.

Traction force microscopy (TFM) is a well-established technique for quantifying the forces that cells exert on their underlying substrates. However, its application to dynamically beating cells-such as cardiomyocytes (CMs) cultured as two-dimensional monolayers-remains challenging, particularly when the cells are grown on non-planar or micropatterned substrates. In this study, we present an integrated TFM-finite element analysis (FEA) workflow integrated with dual-plane fluorescence imaging. This approach enables quantification of the stress and strain energy density (SED) fields generated by human-induced pluripotent stem cell-derived CMs (hiPSC-CMs) cultured on micropatterned polydimethylsiloxane (PDMS) substrates with tunable stiffness. Substrate stiffness was tuned to mimic both healthy (∼5 kPa) and fibrotic (∼50 kPa) cardiac microenvironments. Displacement fields captured from the top and bottom planes of the micropatterns were interpolated and mapped onto a finite element model to reconstruct local stress and strain energy distributions. Results showed that substrate stiffness and micropatterning synergistically modulate cardiomyocyte contractility. Micropatterning promoted cellular alignment and directional force transmission, resulting in anisotropic stress fields and increased SED, particularly on stiff substrates. Moreover, proteomic data revealed a shift from oxidative phosphorylation to glycolysis in cells cultured on stiff micropatterned substrates, consistent with pathological cardiac remodeling. Collectively, these findings demonstrate that soft micropatterned substrates recreate a physiological cardiac microenvironment that supports oxidative metabolism and efficient contractility, whereas stiff micropatterned substrates mimic cardiac remodeling characterized by enhanced stress generation and glycolytic metabolism. The proposed TFM-FEA platform provides a robust and quantitative framework for studying cardiomyocyte mechanobiology under physiologically relevant conditions and can be readily applied to cardiac tissue engineering, disease modeling, and drug screening.