Cells can respond to vibrational frequencies beyond physiological frequency ranges, including those in the ultrasonic domain. Independent, standardized experiments that probe mechanotransduction across frequencies and vibration modalities could clarify the underlying mechanisms that enable this broadband responsiveness.

Barriers in the human body play a crucial role in regulating the exchange of substances between compartments, with permeability alterations occurring under both physiological and pathological conditions. In vitro barrier models are essential tools for studying the mechanisms of molecular diffusion across these barriers. Traditional coculture systems or advanced organ-on-chip (OoC) platforms mostly utilize permeable membranes based on artificial, nonbiodegradable materials. In this study, we introduced cellulose nanofibrils (CNFs)-based membranes to develop a new class of in vitro barrier systems. CNFs, derived from natural sources, are nontoxic, biodegradable, optically transparent, and feature a 3D fibrillar structure that mimics the cellular basement membrane. We successfully modulated the permeability of CNF-based membranes, interposed in dual-chamber polydimethylsiloxane devices, to small molecules through chemical and enzymatic treatments, while preserving their ability to allow cell adhesion and growth. This technology holds potential for its integration in next-generation OoC devices, offering more realistic and complex models that closely mimic the physiological behavior of human barriers.

Enthesis tissue engineering aims to develop scaffolds that replicate the mechanical and structural gradients of the tendon/ligament-bone interface. Among biofabrication techniques, electrospinning is one of the most promising to fabricate morpho-mechanically relevant enthesis fascicle-inspired scaffolds. An unexplored characteristic of these nanofibrous scaffolds is their ability, when mechanically tested, to produce/transmit strain rate and nanofiber-fracture dependent mechanical vibrations, which can potentially influence surrounding tissues and cells. This study develops a method to investigate how scaffold geometry and material affect vibrational behavior under mechanical stimulation. Electrospun bundles of poly(L-lactic) acid/collagen type I (PLLA/Coll) were fabricated to mimic the fibrocartilage, enthesis junction, and tendon/ligament regions, while block copolymer poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT-PBT) bundles represented only the tendon/ligament. Scaffolds were morphologically and mechanically characterized, including strain rate-dependent vibrational response and local mechanical properties via AFM nanoindentation. Scanning electron microscopy confirmed distinct fiber architectures. Under monotonic tensile tests, scaffolds exhibited strain rate-dependent mechanical behavior, PLLA/Coll bundles showed dominant vibrational frequencies up to 4.2 ± 0.9 Hz in a geometry-dependent manner, while PEOT-PBT scaffolds displayed higher vibration attenuation, with dominant frequencies peaking at 0.539 ± 0.063 Hz and lower tensile properties. Nanoindentation revealed spatial gradients in elastic modulus and energy dissipation across PLLA/Coll bundles, supporting a local mechanical decoupling at the enthesis-inspired junction. Integrating vibrational characterization with multiscale mechanical analysis provides a framework for designing scaffolds that more accurately reproduce the gradient mechanical environment of fibrous musculoskeletal tissues. These findings highlight the potential of this combined approach to improve the mechanical comprehension of electrospun scaffolds.

Spheroids have emerged as valuable tools in bone tissue engineering, mimicking the cellular interactions in native tissues. However, the application of small and low-cell-number spheroids for simultaneous bone regeneration and vascularization remains underexplored. In this study, small pre-vascularized spheroids (250 cells each) were developed, using human mesenchymal stem cells (hMSCs) or human periosteum-derived stem cells (hPDCs), co-cultured with human umbilical vein endothelial cells (HUVECs). Spheroids were evaluated for stability, osteogenic differentiation, and angiogenic potential. Results indicated that hMSC and hPDC spheroids formed stable structures, while HUVEC monocultures failed to achieve spheroid stability. Co-cultures showed HUVEC localization patterns mimicking native vascular structures. Gene and protein analyses revealed distinct osteogenic potential between hMSC and hPDC spheroids, with the latter demonstrating superior and earlier differentiation. Additionally, vascular endothelial growth factor expression was higher in co-cultures, suggesting enhanced angiogenic potential, particularly in hPDC spheroids. Using small-diameter spheroids addresses limitations of conventional large spheroids, such as necrotic core formation and heterogeneous differentiation. These findings emphasize the promise of pre-vascularized spheroids for scaffold-free and scaffold-based tissue engineering applications. Furthermore, their small size enables the exploration of their potential applications in 3D bioprinting, paving the way for the future development of more biomimetic vascularized bone constructs.

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 enthesis, the point where a tendon or ligament attaches to bone, is a graded fibrocartilaginous interface that poorly regenerates after injury. Here, we present a modular and scaffold-free strategy for engineering microtissues relevant to enthesis repair by fusing anterior cruciate ligament-derived spheroids with spheroids from osteogenically differentiated human mesenchymal stromal cells. We show that the maturation state of the constituent spheroids governs fusion dynamics and spatial organization, enabling the controlled formation of ligament-, fibrocartilage-, and bone-like regions within a single, radially/concentrically organized construct. Within 10 days, the fused tissues display locally distributed lineage-specific markers and type X collagen localized at the interface between the osteogenically-derived core and ligamentous shell. The latter is indicative of the de novo formation of a fibrocartilage-like region between these regions. The system is scalable by simply adjusting the spheroid number. It supports external mechanical stimulation via ultrasound. The acoustic cues further promote extracellular matrix deposition and tissue growth while maintaining structural integrity. This readily implementable heterotypic spheroid platform offers an in vitro model for studying enthesis mechanobiology, screening therapeutic compounds, and evaluating microscale biomaterials, with translational potential as injectable or bioprintable building blocks for enthesis repair.

The endocrine system is essential for correct human physiology and controls the ability of the human body to develop, grow, and reproduce through the synthesis and release of hormones. In recent decades, numerous studies have highlighted the presence of chemicals that are able to interfere with and damage the functionality of the endocrine system. These compounds have been identified as endocrine disruptors (EDs), and they can have serious effects on human health. Despite this knowledge, functional endocrine gland in vitro models that can be used for the study of endocrine gland development and disease progression and for the evaluation of potential EDs in vitro are lacking. In this review, we provide an overview of the major components of the endocrine system, as well as of the most well-known EDs and their mode of action. With a focus on the thyroid gland, the largest endocrine gland of the body, we report on the current in vitro models available for the evaluation of ED toxicity and mode of action. Finally, we describe new technologies that have been employed for the production of innovative in vitro models.

Reactive oxygen species (ROS) are key regulators of neuronal physiology but contribute to oxidative damage when dysregulated, as in traumatic, ischemic, and inflammatory conditions. Biomaterials capable of replicating the mechanical characteristics of brain extracellular matrix while modulating oxidative stress are therefore of significant interest for neural tissue engineering and in vitro disease modeling. In this study, we functionalized dynamic hyaluronic acid (Ha) hydrogels with 3-aminomethyl phenylboronic acid (PBA) and crosslinked with poly(vinyl alcohol) (PVA) via reversible boronic ester bonds to develop ROS-responsive scaffolds. By varying the degree of PBA grafting, we observed linked feedback governed the mechano-redox properties of Ha-based dynamic hydrogels with the functionalization degree, enabling simultaneous tuning of stiffness, viscoelastic behavior, and antioxidant activity. The developed materials provide a platform for investigating cell responses to mechanically and chemically defined microenvironments and may be useful for modeling oxidative stress-related neuropathological conditions.

Abstract Endocrine disruptors (EDs) are an exogenous group of compounds associated with thyroid malfunctioning in the human body. Nonetheless, there are currently no adequate in vivo or in vitro models for the preclinical testing of these compounds since both animal and two-dimensional (2D) cell-based models are not able to mimic thyroid physiological conditions from both functional and three-dimensional (3D) organization perspective. Recently, bioprinting technologies emerged as an innovative tool in the field of regenerative medicine and advanced 3D in vitro models that allow the creation of 3D well-organized structures able to mirror physiologically relevant tissue and organ architectures. In this study, we evaluated microfluidic bioprinting as a biofabrication technology to develop a 3D in vitro model of the thyroid gland. We studied the fundamental parameters to obtain a fine control over the bioprinted fibres for different biomaterials. Then, we assessed the possibility to bioprint single thyroid cells, thyroid spheroids and finally mouse embryonic stem cell-derived thyroid follicles. The different cell types maintained high viability and metabolic activity. The bioprinted thyroid model showed high expression of different early and late functional markers and to be responsive to ED exposure. These bioprinted thyroid constructs could provide a new set of advanced 3D in vitro models to test potential EDs and possible adverse outcomes that may be associated with their administration or exposure.

In this proof-of-concept study, we develop a novel 3D cell patterning and culture platform. The "Floor-Ceiling-Chip" (FC-Chip) simply consists of two opposing 2D substrates in the form of ion track-etched membranes, creating a pseudo-3D microenvironment for the cells between them. This allows the stimulation of both the dorsal and ventral sides of cells, thereby also eliminating the artificial polarization, for example, of stromal cells, observed in standard culture dishes and inserts. By providing the membranes with micropatterned cell-adhesive islands of varying geometries and sizes, the FC-Chip enables control over cell shape and alignment in a 3D environment. Analysis of fluorescence microscopic images reveals distinct cellular and nuclear morphology, along with perinuclear actin organization, in the on-chip cultures compared to cultures on traditional 2D substrates. Cells in the FC-Chip exhibit fewer focal adhesions, lower expression of lamin A/C, and less nuclear localization of the yes-associated protein 1. The chip demonstrates compatibility with standard biochemical assays and supports long-term cultures up to 10 days, expanding its potential applications. Overall, the early version of the FC-Chip presented here confirms the feasibility of a straightforward, accessible, and versatile future culture platform for the manipulation and modeling of cell morphology and organization in 3D.

The fibrous, viscoelastic extracellular matrix (ECM) directs cell fate through mechanotransduction, but recreating these time-dependent mechanics in biomaterials remains a significant challenge. Current synthetic matrices rarely reconcile fibrillar architecture, physiological stiffness, and stress relaxation, with most systems achieving only some of these hallmarks. Supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogels offer a compelling route forward, as their hydrogen-bonded nanofibers mimic ECM-like networks. Simultaneously, the reversible dynamic hydrogen bonding responsible for the assemblies enables shear thinning, self-healing, and tunable viscoelasticity. Here, three distinct BTA hydrogels were developed, distinguishable by the hydrophilic poly-(ethylene) glycol (PEG) linker length, and all hydrogelators self-assemble and form self-healing, shear thinning hydrogels. Curiously, in contrast to covalent networks, shortening the length of PEG leads to a decrease in stiffness (G') and faster stress relaxation time scales (t 1/2). Blending BTA hydrogelators with two different molar masses leads to an almost linear increase in G' yet a more modest increase in t 1/2. The hydrogels were 3D printed with good shape fidelity, and all three hydrogels are adherent, leading to a self-sustaining construct composed of three regions with distinct G' and t 1/2. These findings emphasize the power of using polymer length as an orthogonal design handle, further expanding our chemical toolbox for developing processable biomaterials with tunable viscoelasticity.

The encapsulation of kidney organoids within hydrogels provides a biomimetic environment that enhances their structural and functional relevance for disease modeling and drug screening. However, the presence of hydrogel matrices poses a major challenge for molecular analysis, particularly for RNA extraction, where residual material can interfere with yield, purity, and downstream applications. The objective of this study was to systematically evaluate RNA extraction methods for kidney organoids encapsulated in alginate-norbornene hydrogels and identify an optimized protocol suitable for reliable gene expression analysis. We compared commonly used extraction methods designed for mammalian tissues and plant-derived materials, with and without prior enzymatic digestion of the hydrogel. RNA yield and purity were assessed by spectrophotometry and fluorometry, while RNA integrity was analyzed by Bioanalyzer, and performance in downstream assays was evaluated by quantitative PCR of housekeeping genes. Our results showed that RNA yield was consistently lower in encapsulated organoids compared to suspension cultures, reflecting smaller organoid size and reduced metabolic activity in encapsulated conditions. Spectrophotometric purity ratios differed between suspension and encapsulated samples, but RNA integrity was preserved across all methods, with values within the acceptable range. Quantitative PCR revealed that TRIzol-based extractions introduced significant variability between suspension and encapsulated samples. Conversely, the protocol with alginate lyase digestion followed by the Maxwell RSC RNA kit produced the most reproducible results. Ct values for control and encapsulated samples were highly consistent, with inter-condition variability remaining below 0.5 standard deviations across replicates. These findings highlight the importance of adapting RNA isolation protocols to account for the presence of hydrogels. Alginate lyase digestion combined with a plant RNA extraction kit offers a reliable strategy for obtaining high-quality RNA from encapsulated kidney organoids within alginate-based hydrogels. While this approach enabled a robust gene expression analysis providing a foundation for transcriptomic studies, different hydrogels and organoid combinations might require additional adjustments underscoring the importance of adopting methods to ensure optimal RNA quality for downstream methods.

Human induced pluripotent stem cells (hiPSCs)-derived kidney organoids can resemble early stages of human kidney development, morphology and architecture. However, one of the main limitations of the organoids is the reduced vascularization, which limits differentiation and maturation. To increase the oxygen and nutrient supply, multiple vascularization strategies were proposed in literature, including organ-on-chip, hydrogels with angiogenetic cues, and co-culture with endothelial cells. In this work, we developed a three-dimensional (3D) printed chip by extruding sacrificial pluronic, in a fully automated and cost-effective way. By dissolving the pluronic, two circular cross-sectional channels, together with three separated central gel compartments, were created. Human umbilical vein endothelial cells (HUVECs) were seeded in the coated 3D printed chip, and after seven days kidney organoids were added in the central gel compartments, embedded in a partially digested decellularized extracellular matrix (ddECM) hydrogel, and co-cultured for five days under perfusion. At the end of the co-culture, capillary-like structures were formed towards the organoids both in the outer and central parts, colocalizing with LTL and PODXL positive stained areas. We were able to develop primitive capillary-like structures throughout the organoids, using an ad-hoc designed 3D printed chip. Our strategy provides new possibilities to investigate further organoid maturation, drug testing and disease modeling.

Titanium is widely used in orthopedic and cranio-maxillofacial surgery because of its excellent mechanical properties and biocompatibility, but it lacks bioactivity in bone. Incorporating calcium phosphate ceramics such as β-tricalcium phosphate (TCP) can enhance osteoconductivity. Additive manufacturing methods based on powder bed fusion typically cannot produce metal-ceramic composites. This study evaluated human mesenchymal stromal cell (hMSC) responses to novel 3D porous titanium-alloy (Ti6Al4V) scaffolds fabricated by 3D fiber deposition containing up to 10 wt% TCP. Scaffolds were manufactured as Ti6Al4V alone (Ti0) or composites with 5 wt% (Ti5) or 10 wt% (Ti10) TCP, with ∼500 μm fully interconnected pores. hMSCs were cultured for up to 28 days in basic, osteogenic, or mineralization medium. Cell behavior was assessed by DNA and metabolic assays, osteogenic differentiation by ALP activity, ELISA (OCN, OPN) and RT-qPCR (RUNX-2, ALP, OCN, OPN), ECM formation by SEM, and mineralization by Alizarin Red S staining. All scaffolds supported cell attachment and metabolic activity. Early osteogenic markers were reduced in TCP-containing scaffolds, whereas late-stage markers (OCN, OPN) were upregulated in Ti10. ECM coverage was complete after 14 days. Mineralization showed an increasing trend with increasing TCP content. These 3D porous Ti6Al4V-TCP scaffolds support hMSC attachment, osteogenic differentiation, ECM formation, and mineralization in vitro, suggesting potential for regeneration of large load-bearing bone defects.

Endocrine disrupting chemicals (EDCs) are ubiquitous environmental contaminants capable of dysregulating the production of thyroid hormones. Traditional thyroid toxicological assays rely on 2D cell cultures and animal models, both of which fail to accurately recapitulate human thyroid physiology and provide limited mechanistic insight into EDC toxicity. To overcome these limitations, we report a novel thyroid-on-chip platform integrating mouse embryonic stem cell–derived thyroid organoids with advanced organ-on-chip (OoC) technology and downstream multi-omics analysis. The platform leverages a reversibly-sealed microphysiological flow battery (MFB) to allow scale up of dynamic organoid culture and controlled chemical exposure while reducing operational complexity. Upon EDC exposure, transcriptomic and proteomic analysis revealed new molecular signatures of thyroid disruption across four different EDC classes, even at very low EDC concentrations (1nM), validating the capacity of this system to mechanistically dissect EDC-induced responses. This represents an integrated platform consists of an advanced physiologically relevant assay framework for next-generation endocrine toxicity testing, bridging the gap between in vitro screening and in vivo thyroid physiology.

It has been reported that cells need a more physiologically relevant micro-environment that allows them to maintain their phenotype. When cultured on 2D tissue culture plates, human mesenchymal stem cells (hMSCs) lose their differentiation capacity and clinical potential. Here, we developed a 3D alginate hydrogel functionalized with the Arg-Gly-Asp (RGD) sequence and having mechanical stiffness mimicking the mechanical properties (<5 kPa) of bone marrow. hMSCs cultured in these hydrogels were halted in G(1) phase of the cell cycle and non-proliferative, as shown by flow cytometry and 5-Ethynyl-2'-deoxyuridine (EdU) staining, respectively. Their quiescent state was characterized by an upregulation of enhancer of zeste homolog 1 (EZH1) at the gene level, forkhead box O3 (FoxO3) and cyclin-dependent kinase inhibitor 1B (p27) at the gene and protein levels compared to hMSCs grown in 2D. Studies in 3D hydrogels of collagen or alginate-RGD hydrogels presenting a higher concentration of the peptide revealed that, independently of the concentration of RGD or the chemistry of the adhesion motives, hMSCs cultured in 3D presented a similar phenotype. This phenotype was exclusive to 3D cultures. In 2D, even when cells were serum-deprived and became non-proliferative, the expression of these markers was not observed. We propose that this difference may be the result of mammalian target of rapamycin complex 1 (mTORC1) being downregulated in hMSCs cultured in 3D hydrogels, which induces cells in "deep" quiescence. Our results represent a step forward towards understanding hMSCs quiescence and its molecular pathways, providing more insight for hMSCs cell therapies.

Non-sulfated polysaccharides like hyaluronic acid (HA) have been widely studied as scaffold material for tissue engineering applications. To mimic the function of sulfated glycosaminoglycan in the matrix, sulfate groups can be grafted. However, here, harsh reaction conditions are required which induce significant backbone degradation. As an alternative, sulfonates (R-SO3 -) have been shown to resemble the function of sulfates yet have not been introduced on polysaccharides. Using a two-step strategy, we introduced a tunable amount of sulfonate groups on HA, without requiring harsh reaction conditions and organic solvents. By varying the degree of carboxylic acid activation using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), norbornene (NB, 3-18%) or maleimide (MAL, 2-14%) groups were grafted. Subsequently, 3-mercapto-1-propanesulfonate was coupled in high efficiency on the addressable groups via orthogonal thiol-ene and thiol-Michael addition. Additionally, we demonstrated the formation of hydrogels using poly(ethylene glycol)-di-SH as a crosslinker. However, because of the low crosslinking kinetics, HA-MAL appeared not useful for application. Simultaneous addition of the crosslinker and MPS to norbornyl-conjugated HA's in various ratios enabled the formation of hydrogels with tunable stiffness and degree of sulfonate groups. The simple strategy is likely applicable to other commonly used polysaccharides and therefore interesting to the broader tissue engineering community.

Autologous grafts remain the clinical gold standard for vascular reconstruction; however, their use is limited by donor site morbidity, poor availability, and long-term failure. Synthetic alternatives, while effective in large-caliber vessels, fail in small-diameter applications (<6 mm) due to thrombosis, intimal hyperplasia, and biomechanical mismatch. In this context, tissue-engineered vascular grafts (TEVGs) emerge as a solution, requiring biomaterials that closely replicate the structural, mechanical, and hemocompatible properties of native vessels. Aliphatic polyesters such as polylactic acid, polyglycolic acid, and poly(ε-caprolactone) are extensively studied but show poor endothelialization and mechanical deficiency. In contrast, poly(butylene trans-1,4-cyclohexanedicarboxylate) (PBCE) attracts interest for its biocompatibility, thermal stability, and processability. Its copolymerization with Pripol 1009, a commercial fatty diacid, enables modulation of mechanical properties and degradation rate, two of the key parameters for vascular engineering. In this work, electrospun scaffolds based on these copolymers are fabricated in flat and tubular formats and characterized in terms of morphology, mechanical behavior, hemocompatibility, and endothelialization potential. Certain formulations display mechanical properties comparable to native vessels, support endothelialization and smooth muscle cell adhesion, and do not trigger coagulation pathways in in vitro assays. These results identify PBCE/Pripol copolymers as promising candidates for next-generation TEVGs, bridging the gap between synthetic reliability and biological performance in small-diameter vascular applications.

Human-derived biomaterials offer several advantages over animal-derived or synthetic alternatives, including improved biocompatibility, ethical acceptability, sustainability, and clinical translatability. Here we present new applications of human placenta-derived materials - specifically HUMAN PLACENTA substrate, collagen type-I, and Laminin-111 - as 2D coating materials and 3D matrices for the cultivation of spheroids and adherent cells. Collagen type-I coatings supported colorectal cancer spheroid formation without the need for growth-factor supplementation. Lm-111 significantly enhanced NIH3T3 fibroblast adhesion compared with poly-L-lysine and rat-tail collagen type-I, performing comparably to bovine fibronectin. In a transwell blood-brain barrier model, HUMAN PLACENTA substrate coatings enabled confluent endothelial monolayers with transendothelial electrical resistance values not significantly different from the conventional human collagen type-IV/bovine fibronectin mixture. Across these in vitro models, placenta-derived materials performed comparably or better than conventional animal-derived and synthetic coatings, supporting robust cell viability, adhesion, and barrier formation. Due to their human origin, these biomaterials exhibit reduced biological complexity while enhancing biocompatibility and translational relevance. Therefore, they provide a sustainable, ethically acceptable alternative for advanced cell culture systems.