Biofabrication最新文献

Bioprinting is a tissue engineering approach which has great potential to devise regenerative therapies and alleviate tissue/organ shortage.In situbioprinting, allowing the direct creation of functional 3D tissues/organs at the defect site in the patient body, has attracted significant attention of surgeons and researchers. However, it is challenging to design adequate combinations of manufacturing devices, bioinks and cells to meet thein situbioprinting requirements of medical functions, complex human body environment and clinical applications. This review highlights current state-of-art bioprinting technologies, summarising their advantages and challenges forin situapplications from four perspectives: bioprinting methods, bioinks, cell sources and advanced bioprinting strategies.

Osteosarcoma (OS) is the most common primary bone malignancy affecting children and adolescents, for which survival has not improved in more than four decades. The lack of accurate OS preclinical models hinders the understanding of tumor heterogeneity and its interaction with the surrounding extracellular matrix (ECM), limiting the discovery of predictive biomarkers and the development of effective therapies. Four 2D preclinical models from OS patients were established and characterized for their functional differences in comparison to OS cell lines for their growth, cellular phenotypic attributes, osteogenic differentiation capabilities and metabolic responses to growth factors and BMP-2. Molecular and cellular profiling revealed intra-tumoral heterogeneities that were very distinct from the endorsed OS cell lines. The OS patient-derived (PD) cells also displayed differential sensitivity to Doxorubicin and Cisplatin and resistance against Methotrexate. Subsequently, the 3D PDTs (Patient- derived tumoroids) models were developed by self-aggregating spheroids with and without Matrigel® ECM matrix. These PDTs models were screened for selective ECM and bone-specific gene markers, revealing dynamic differences between 2D and 3D models with and without ECM, with heightened dysregulation observed in 3D systems. The drug response variances observed among 2D OS cells and 3D tumoroids model within Matrigel® highlights the need for optimized platforms forin vitropersonalized drug screening. Thus, our findings support the screening of preclinical PD OS models for phenotypic profiling and elucidating ECM contributions to drug responses and pathophysiology.

Intervertebral disc (IVD) degeneration is the primary contributor to low back pain, the leading cause of disability worldwide. Although various triggers have been associated with IVD degeneration, its precise aetiology remains unclear. Consequently, current treatments fail to address the underlying degradative processes. Mechanical loading plays a critical role in IVD homeostasis, and aberrant mechanical stimulation has been identified as a key driver of extracellular matrix degradation in the proteoglycan-rich core of the IVD-the nucleus pulposus (NPs). Elucidating the molecular mechanisms of IVD mechanotransduction could therefore be pivotal in identifying effective drug targets. However, we are lacking easy-to-use, reliable models to study IVD's mechanobiological mechanisms in human cells. Here, we present the first mechanically active, microscale, human cell-based NP-on-a-Chip (NPoC) model that mimics the native NP microenvironment and enables controlled investigation of mechanically induced degenerative processes. Starting from primary human NP cells, we demonstrate that hypoxic culture (i.e. 2% O₂) results in 3D constructs with gene expression levels of NP markers (ACAN, COL2A1, CDH2, OVOS2), and matrix composition (collagen type II and glycosaminoglycans) comparable with the native NP tissue. NPoC constructs respond to cyclic compression in an intensity- and duration-dependent manner. Physiological compression (10%) enhances glycosaminoglycan deposition, whereas hyperphysiological compression (30%), especially if prolonged in time (16 h d^-1), induces upregulation of inflammatory and catabolic markers (PTGS2, MMP13), matrix degradation, and increased apoptosis-thus recapitulating clinical hallmarks of NP degeneration. As a proof of concept for the platform's perspective utility in therapeutic screening, we demonstrate that inhibition of the mechanoresponsive channel TRPV4 with GSK205 restores baseline expression levels of mechanosensitive and catabolic genes. The new NPoC is thus suitable for studying NP mechanobiology and screening mechanotransduction-targeting drugs, and it may facilitate the future discovery of disease modifying therapies for discogenic low back pain.

Force generation dynamics in native muscle tissues have been stringently optimized by evolution. Realizing similar contractile dynamics in a widely available biomaterial and subsequently fabricating macroscopic functional modules from them remains challenging. Herein, we tailor two-photon stereolithography to 3D print synthetic muscles made from bovine serum albumin to realize 1 mm long contractile fibers. We show that pH-dependent contractions in these synthetic muscles follow parabolic force-length relationships similar to biological muscles. Achieved stress outputs of 0.78 ± 0.13 N/cm2 were within an order of magnitude of smooth and cardiac muscle. Stretch-shortening work loops performed under different strain rates in turn revealed a viscoelastic behavior and significant velocity dependence of work and net power, more similar to skeletal muscle. That an isotropic protein hydrogel can achieve such dynamics, reinforces the notion that these are not limited to sarcomere-level ordering and suggests a more general design space for non-canonical conformational dynamics to engineer performance improvements in artificial muscle materials.

The investigation of breast cancer initiation and progression has been significantly advanced by the development of three-dimensional (3D) in vitro models, which provide a more physiologically relevant representation of the tumor microenvironment (TME) compared to conventional two-dimensional cultures. Over the past decade, and particularly since 2020, a wide range of strategies has been developed to generate stable and functional 3D breast cancer models. This review provides a comprehensive overview of the most promising (bio)fabrication-based technologies developed for breast cancer modeling, critically discussing their applications, advantages, limitations, and future perspectives. Among current approaches, tumor spheroids have demonstrated considerable value due to their characteristic architecture, comprising a necrotic core surrounded by proliferative and quiescent cell layers, which partially mimics in vivo tumor organization. In parallel, organ-on-chip (OoC) systems have emerged as powerful platforms for drug screening and therapeutic testing, enabling dynamic culture conditions within microengineered and perfusable environments. However, spheroids lack an external extracellular matrix, while the compartmentalized nature of OoCs systems limits their ability to fully reproduce the structural and compositional complexity of the breast TME. To address these limitations, engineered 3D-printed scaffolds and constructs produced through biofabrication approaches have gained increasing attention. In particular, natural hydrogel-based systems offer high biocompatibility and tunable biochemical and mechanical properties, enabling the co-culture of malignant and healthy cells and supporting more predictive evaluations of anticancer therapies.

Multi-nozzle (MN) collaborative bioprinting enables high-precision fabrication of complex tissue and organ models through synchronous deposition of heterogeneous bioinks within a shared substrate, offering a promising solution for efficient construct generation. However, challenges remain, including nozzle motion interference and inconsistent geometric fidelity when printing asymmetric structures with heterogeneous materials. This study proposes a multi-nozzle collaborative and alternating printing path (MN-CAPP) planning strategy that integrates intra-layer repartitioning with adaptive mode switching to optimize the fabrication of complex heterogeneous tissues. By printing two Y-shaped vascular models with distinct interfaces, MN-CAPP preserves the efficiency advantages of collaborative printing for symmetric regions, improving printing efficiency by 32.4% and 33.0%, respectively, compared with single-nozzle printing. Furthermore, MN-CAPP adaptively regulates printing strategies for regions with significant nozzle step differences based on ink rheology and printing parameters. During the fabrication of size-differentiated scaffolds, the proposed path effectively suppresses edge material stack in small-scale scaffolds, resulting in a 33.8% improvement in pore diffusion degree relative to conventional collaborative printing. Finally, successful fabrication of a heterogeneous rabbit hepatobiliary model demonstrates a deviation of ⩽4% in critical feature dimensions from design specifications, confirming MN-CAPP's effectiveness in enhancing both printing precision and dimensional reproducibility for complex asymmetric structures.

Microtissue-based strategies have gained significant attention for the fabrication of cartilage grafts. Their spatial organization within three-dimensional constructs plays a crucial role in directing tissue formation and maintaining the immediate mechanical stability of the printed structure. Melt-electrowritten (MEW) fibrous scaffolds have been widely used to reinforce cell-laden hydrogels, while also guiding microtissue fusion and self-organization within the constructs. However, current bioprinting methods used for positioning microtissues or spheroids within these structures are limited by insufficient control over spheroid deposition, low throughput, and technical challenges, such as nozzle-clogging. In this study, we leveraged laser-induced forward transfer (LIFT) to print articular cartilage progenitor cell (ACPC) spheroids of two different sizes (Ø ∼80 and 150μm) into fibrous polycaprolactone scaffolds. We investigated the effects of laser energy, spheroid size, and concentration in the bioink to identify the key parameters for controlled deposition. Furthermore, we assessed print fidelity, post-print spheroid viability, and chondrogenic differentiation capacity. The deposition rate of the spheroids was studied to maximize transfer efficiency, and the resulting optimal parameters were subsequently applied to place the spheroids within the MEW meshes. However, the spheroid transfer efficiency remained limited, not due to shortcomings in the printing process, but because uniform encapsulation becomes challenging when working with discrete and larger entities, such as spheroids. While single cells benefit from homogeneous suspension, enabling random encapsulation, spheroids require precise targeting to be successfully transferred. To address this challenge, an AI-based imaging analysis system was employed, and the amount of bioink on the donor slide was reduced to improve the transfer of larger spheroids further. Here, we demonstrate for the first time the successful convergence between LIFT and MEW for the deposition of ACPC spheroids into reinforcing meshes as the next step towards automated production of tissue constructs.

The myotendinous junction (MTJ) is a critical interface connecting skeletal muscle and tendon, responsible for transmitting contractile forces and ultimately enabling musculoskeletal movement. Due to its complex architecture, the MTJ is particularly susceptible to injury under conditions of excessive stretching, high-impact loading, aging and neuromuscular disorders such as muscular dystrophies. Despite its significant physiological role, research on the MTJ remains limited, primarily due to the challenges associated with obtaining human tissue samples. This limitation underscores the urgent need for advancedin vitromodels that can accurately replicate tissue-specific features. In this work, we developed a human-derived 3D MTJ-like model using the rotary wet-spinning technology. Human primary pericytes and human tendon derived stem cells were spatially patterned within the extruded hydrogel fibers in a consecutive manner to form highly integrated and anisotropically aligned biomimetic multicellular tissue constructs. Upon maturation, immunofluorescence analysis confirmed the presence of tendon and muscle-tissue specific markers including collagen type I, collagen type III, tenascin, tenomodulin and myosin heavy chain, respectively. Specifically, cellular organization recapitulated the interdigitated architecture typical of the MTJ native microenvironment. Moreover, the expression of collagen type VI, thrombospondin 4, and collagen type XXII, along with the polarized localization of paxillin and neural cell adhesion molecule 1 at the myotube-tendon interface, confirmed the establishment of a highly specialized junctional niche characterized by active cell-matrix interactions and cytoskeletal anchorage. Collectively, our biomimetic 3D model could offer a promising platform for the in-depth investigation of musculoskeletal development, pathophysiological processes, and the advancement of targeted therapeutic strategies.

Three-dimensionalin vitromodels are critical for recapitulating key aspects of neural network development and interregional interactions. We present a scaffold-free modular system based on primary cortical and hippocampal neurospheroids (NSs), which are subsequently coupled to self-assemble into reproducible assembloid-like structures (ASs). Through a multimodal approach, we characterized their morphological, mechanical, metabolic, and functional properties. NSs displayed progressive growth, viability surpassing 2D cultures, and stiffness approaching physiological brain ranges. Immunostaining verified proper neuronal and astrocytic ratios and confirmed a physiologically relevant GABAergic component. Upon coupling, ASs exhibited robust structural integration while maintaining functional modularity. Calcium imaging enabled the investigation of synchronization patterns at modules' interface, while electrophysiology revealed maturation-dependent and configuration-specific emergence of rhythms, a type of activity typically foundin vivo. Functional excitation-inhibition balance remained constant throughout development and was pharmacologically modulated successfully. Our platform balances biological relevance and experimental tractability, offering a versatile tool for investigating neural circuit development, network dynamics, and region-specific perturbations in a reproducible and scalablein vitroenvironment.

Despite decades of advancements in melt electrowriting (MEW) and electrospinning (ES), poly(ϵ-caprolactone) (PCL) remains the gold standard polymer for these techniques. Its widespread use is attributed to its processability in both melt and solution, thermal stability, and low melting temperature. MEW and ES enable the fabrication of micro- and nanofibrous scaffolds that can mimic the extracellular matrix of specific tissues. This makes them especially attractive for biomedical applications. However, PCL lacks key properties, e.g., elasticity and suitable biodegradation time, for soft tissue applications. We report for the first time the successful introduction ofα-amino acid-based poly(ester amide)s (AAA-PEAs) for MEW processing. Additionally, ES outcomes have been improved compared to existing literature. These polymers were synthesized via polycondensation containing ester and amide functionalities, yielding high molar masses (>40 kg mol^-1). L-alanine and L-phenylalanine were selected asα-amino acids, differing in hydrophobic side groups (methyl vs benzyl), resulting in distinct material properties. Both AAA-PEAs showed excellent ES processability, producing uniform fibers (1-3μm) without the need of adding PCL. Notably, only the phenylalanine-based PEA was so far processable by MEW, yielding smooth, uniform fibers (25-30μm) with no pulsing, fusing, or surface defects. As proof-of-concept, excellent stacking behavior up to 15 layers was achieved. Scaffolds exhibited enhanced ultimate tensile strength (13.70 ± 1.60 MPa) and accelerated biodegradation (72%-77% remaining mass after 16 weeks in phosphate buffered saline at 37 °C) compared to PCL.In vitrostudies with MC3T3-E1 cells confirmed cytocompatibility. The above findings underscore the potential of AAA-PEAs as promising biomaterials for soft tissue biomedical applications.