Biofabrication最新文献

Among additive manufacturing (AM), 3D inkjet technology, materials extrusion (ME), and digital light processing (DLP), which are from dot and line to face printing, have been extensively investigated for biological and pharmaceutical applications. These techniques are valued for their ability to create customised, complex, drug-laden devices and tissue engineering scaffolds. However, testing new bioinks or filament designs can be both expensive and time-consuming. To this end, numerical simulation offers a useful solution by reducing costs and saving time. Both machine learning (ML) and theory-based models can be used for simulation. ML excels in handling complex data but faces challenges with data availability and overfitting, while theory-based models provide a more interpretable and data-efficient framework. This review explores how theory-based numerical simulation can be used to assess and optimise factors such as bioink printability, technique mechanism, printing parameters, and post-printing outcomes. By using simulation, key parameters can be understood and optimised without performing extensive physical experiments. The review highlights current models and discusses opportunities and challenges in using simulations to enhance the AM process, potentially advancing regenerative medicine and personalised treatments.

Osteocytes play a major role in the regulation of bone remodelling and homeostatis and should therefore be more prominently incorporated intoin vitrobone models. Bioprinting is a versatile method to generate spatially defined tissue-like structures, however bioprinting of osteocytes remains challenging due to their post-mitotic nature. This study therefore investigates the transition of primary human osteoblasts (hOB) into osteocytes in bioprinted constructs, with respect to bioink composition and growth factor supplementation. Osteocytes were successfully differentiated from hOB, within bioprinted constructs via using alginate/methylcellulose/gelatin ink, alginate/methylcellulose/egg white ink and alginate/methylcellulose/human plasma ink (Alg/MC/Pl). Osteocytic morphology and marker expression was confirmed by fluorescence microscopy and gene expression analysis. Moreover, a significant upregulation of late osteocytic markers (e.g.SOSTandMEPE) was observed under low serum conditions (2%) compared with cultures maintained in 10% fetal calf serum (FCS). Human platelet lysate (hPL), evaluated as an alternative to FCS, also demonstrated the capacity to support osteocyte differentiation. Furthermore, hPL was successfully used for hOB pre-differentiation. In the presence of 2% hPL, a higher number of multinucleated osteoclasts along with an elevated activity of osteoclast-specific enzymes (tartrate-resistant acid phosphatase, cathepsin K and carbonic anhydrase 2) in comparison to 2% FCS was observed. This high potential of hPL to support osteoclastogenesis opens the way for physiologically-relevantin vitrobone models comprising both osteocytes and osteoclasts. Indirect co-cultures of human osteoclasts and human osteocytes, bioprinted and differentiated in Alg/MC/Pl showed expression of all relevant osteoclast and osteocyte markers.

Light-based bioprinting has rapidly expanded as versatile platforms to replicate the complex architectures of native tissues, by allowing spatio-temporal localization of biomaterials and cells. These approaches rely on bioresins composed of photo-crosslinkable polymers, photoinitiators, and, where appropriate, photoabsorbers. In this perspective, we summarize recent technological progress in light-based bioprinting, moving beyond mere structural complexity toward the creation of engineered constructs that recapitulate the native tissue function. We discuss the development of bioresins adapted from a long history of tissue engineering and regenerative medicine research, with an emphasis on shifting the field from structural mimicry toward physiologically relevant biological function. We also highlight current limitations, including the constraints of bioprinting workflow, bioresin compositions, and the need to focus more on downstream cellular signaling and function, rather than just basic cytocompatibility. Finally, we suggest several considerations for next-generation bioresin and printing strategies better tailored for clinical translation, including improved control over cellular microenvironments and standardized, regulatory-accepted and reproducible formulations.

Arteriovenous grafts are widely used for hemodialysis access, yet high failure rates remain a major clinical challenge. Decellularized blood vessels offer promising alternatives, but effective protocols must remove cellular and antigenic components while preserving extracellular matrix (ECM) integrity. This is particularly challenging in elastic arteries, where smooth muscle cells are anchored by dense actin filaments that are difficult to remove without ECM damage from harsh treatments. Here, we present a decellularization strategy based on actin-disrupting agents, specifically Latrunculin B, to facilitate removal of these dense cytoskeletal structures. The protocol integrates osmotic shock, high-ionic-strength salts, and Triton X-100 within a shortened processing time. Full-length porcine carotid arteries were decellularized using a dual-flow perfusion system, and five protocols were evaluated for removal of nuclear and immunogenic material, ECM preservation, mechanical properties, and cytocompatibility with human endothelial cells (HUVECs). All protocols substantially reduced nuclear material while preserving elastin, collagen, and mechanical integrity. Complete removal of immunogenic proteins was achieved with a final alkaline Triton X-100 wash (pH 8-14), highlighting the effectiveness of alkaline treatment in solubilizing membrane-bound antigens. All scaffolds supported HUVEC adhesion and formed a confluent endothelial monolayer within 8 d. Overall, integrating actin disruption via Latrunculin B with dual-salt-Triton processing, alkaline washing, and perfusion enhances decellularization efficiency, preserves ECM structure, reduces processing time and cost, and yields cytocompatible small-diameter vascular scaffolds with strong translational potential.

Melanoma progression, regression, dormancy, and drug resistance involve a dynamic interplay between the tumor mass, dermal and epidermal cells, extracellular matrix (ECM), and the administered therapeutic agent. Understanding the mechanisms behind drug and melanoma interactions as well as the possible collateral effect on the surrounding healthy tissue could improve patient outcomes. To mechanistically unravel these complex interactions in melanoma, there is a need to develop realistic preclinicalin vitromodels; however, current melanoma models fail to replicate not only drug-cell, but also cell-cell, and cell-ECM interactions. Here, a physiologically relevant scaffold-free 3D melanoma model that mimics the morphological and functional features of the melanoma lesion was developed and assessed using an approved therapeutic agent. For this, ECM-coated fibroblasts were assembled with BRAF+ melanoma spheroids to generate the dermis followed by keratinocytes addition and differentiation to form the epidermis. Vemurafenib (Vem), a BRAF inhibitor (BRAFi), was evaluated for its efficacy on 2D melanoma cells, spheroids, and 3D scaffold-free melanoma models. Reduced cellular viability across all models demonstrated the potency of Vem in inhibiting BRAF+ melanoma. In spheroid-only and 3D melanoma skin models, Vem significantly reduced tumor size; however, spheroid-only models exhibited a slightly enhanced tumor shrinkage compared to spheroids embedded within skin models. Analysis of ECM-related genes showed a tendency to be downregulated in melanoma skin compared to healthy skin models, which was partially recovered post-Vem application, indicating significant influence of both the tumor and BRAFi in remodelling of the tumor microenvironment. Collectively, the developed skin model bridges the gap between 2D cultures, conventional spheroids, and complex patient-derived tumors. In future, the developed models can be utilized for personalized drug screenings to enhance translational potential of targeted therapies in multiple skin cancer subtypes.

The development of reliablein vitromodels for stratum corneum (SC) permeation studies remains a significant challenge. Ethical constraints, interspecies disparities, and inter-individual variability in skin underscore the need for standardized skin equivalents with significant research and commercial value. Conventional artificial skin models typically lack the characteristic 'brick-and-mortar' structure of the SC, the primary barrier of the skin, leading to measurable functional deviations from native tissue. To address this gap, we developed a biomimetic SC (ASC) that faithfully replicates the brick-and-mortar structure of the human SC. Polymer microspheres of a size mimicking corneocytes-composed of an optimized ternary polycaprolactone/poly(methyl methacrylate)/polyhydroxybutyrate blend-served as 'bricks' embedded within a lipid-based 'mortar' via a thermally assisted compression molding process. The resulting ASC demonstrated barrier properties comparable to those of excised human epidermis membrane (HEM). Permeability coefficients for five model drugs of diverse properties showed a Pearson's correlation ofr> 0.99 between ASC and HEM. Molecular simulations further elucidated the enhanced intermolecular interactions and restricted drug diffusion within the optimized polymer matrix. Furthermore, the ASC exhibited storage stability, maintaining consistent barrier properties over four weeks at -20 °C. This structurally biomimetic ASC represents a promising, predictive, and animal-free platform forin vitropermeation testing.

Mechanical and geometric cues play a crucial rolein vivo, regulating both morphogenetic processes and proper tissue function. This is particularly evident in skeletal muscle, where aligned architecture is essential for myogenesis and functional force generation. However, precisely engineering tissue geometry at both macroscopic and microscopic scales while simultaneously controlling internal mechanical forces remains a significant challenge. In this study, we introduce a magnetic tissue engineering platform based on a magnetic bioprinting technique, enabling control of biophysical cues that guidein vitrotissue organization. Applied here to skeletal muscle, this approach allows for the rapid fabrication of cohesive tissues in any desired shape using cells labeled with magnetic nanoparticles. Additionally, multiple cell types can be incorporated and spatially organized within the same construct through magnetic segregation. As the tissues tend to transition toward a spherical shape after a few days, their geometry was optimized to further enable magnetic actuation, including the ability to trap and maintain tissue shape over time. Furthermore, this magnetic platform facilitates the investigation of how tissue architecture influences mechanical properties, such as resistance to rupture. Overall, this study highlights the significant potential of magnetic bioprinting and stimulation for controlling tissue morphology and advancing biomechanical research.

Matrigel-based hydrogel microspheres fabricated using microfluidic technology have emerged as promising carriers for tumor organoid modeling. However, their intrinsic low stiffness limits their ability to mimic the mechanical properties of the native tumor microenvironment. To overcome this limitation, we developed an interpenetrating polymer network by incorporating alginate into Matrigel, yielding alginate-Matrigel (AM) composite hydrogel microspheres with tunable stiffness via droplet microfluidics technique. Additionally, to eliminate the cytotoxic effects associated with acidic conditions in alginate-based droplet microfluidics process by acid-driven gelation methods, a novel external gelation strategy was designed. A Ca²⁺preloaded gelatin substrate (contact angle > 80°) was employed to receive AM droplets, enabling rapidin situcrosslinking and solidification. Notably, cells encapsulated within AM microspheres exhibited high viability throughout the gelation process, and the resulting microspheres displayed excellent sphericity and structural uniformity. The entire workflow-spanning droplet formation, allocation, gelation, culture, and drug testing-was integrated into a streamlined single-step process optimized for high-throughput screening. The stiffness significantly increased over 7-fold, elevating from 0.6 kPa in Matrigel microsphere to 5.0 kPa in AM microsphere. Compared to Matrigel-only microspheres, both patient-derived tumor organoids and cell line spheroids in AM microspheres demonstrate enhanced chemoresistance, as indicated by elevated IC₅₀values. Taken together, this simple, biocompatible, and reproducible fabrication strategy offers a powerful platform for organoid modeling, drug screening, and patient-relevant drug testing.

The fibrotic stroma drives tumor progression and impairs therapy, but existing models fail to capture its dual barrier and signaling roles, limiting mechanistic understanding and therapeutic development. To address this, we established a patient-derived fibrotic tumor model that recapitulates key pathophysiological features: extracellular matrix remodeling, substantial tissue stiffening (12-fold increase), and pro-malignant transcriptomic reprogramming. This model demonstrated microenvironment-mediated chemoresistance, increasing the area under the dose-response curve by 1.3-fold and suppressing oxaliplatin-induced apoptosis. Although stiffening delayed drug penetration, barrier disruption did not restore sensitivity. Instead, stiffness inactivated the Hippo pathway, activating YAP as the key chemoprotection driver. YAP inhibition restored drug sensitivity without altering stiffness, revealing a targetable biochemical mechanism independent of the physical barrier. Using this insight, we designed a sequential regimen in which nintedanib preconditioning precedes chemotherapy, achieving near-complete tumor eradication (<5% tumor viability). This work provides a physiological model of stromal biology and a mechanism-guided strategy to overcome microenvironment-mediated chemoresistance.

Blood-brain barrier (BBB) integrity is critical for central nervous system (CNS) homeostasis and represents a key challenge for drug delivery. Three-dimensional (3D) bioprinting offers unprecedented control over architecture and cellular organisation in neurovascular models. This review presents recent advances in bioprinting methodologies, including extrusion-based coaxial nozzle printing for multilayered capillaries, droplet-based drop-on-demand deposition, digital light processing and two-photon polymerization for high-resolution vascular features, and hybrid systems integrating electrospun scaffolds. We detail composite bioink formulations combining natural and synthetic hydrogels that promote endothelial cell viability, tight junction protein expression and shear-responsive barrier function. Main outcomes reveal that 3D printed constructs achieve selective molecular permeability, sustained barrier integrity under perfusion and improved repeatability compared to conventional static cultures. We also discuss emerging strategies such as incorporation of astrocytes and pericytes to replicate the full neurovascular unit, integration of biosensors forin situmonitoring and scalable manufacturing approaches. Addressing current limitations in capillary-scale resolution, mechanical robustness and long-term culture stability will be essential to translate 3D bioprinted BBB models into standardized platforms for CNS drug screening and mechanistic investigations.