Materials Today Bio

Alignment and orientation of cells in vivo plays a crucial role in the functionality of tissue. A challenged faced by traditional cell culture approaches is that the majority of two-dimensional substrates fail to induce a controlled alignment of cells in vitro. To address this challenge, approaches utilizing mechanical stresses, exposure to electrical fields, structurally aligned biomaterials and/or textured microfabricated substrates, have been developed to control the organization of cells through microenvironmental stimuli. In the field of muscle tissue engineering it is often desirable to control the alignment and fusion of muscle precursor cells as it more closely resembles in vivo conditions. In this study, we utilize plant-derived cellulose biomaterials to control the in vitro alignment of C2C12 murine myoblasts. We hereby report that cells display a clear sensitivity to the highly aligned vascular bundle architectures found in decellularized celery (Apium graveolens). Conveniently, the xylem and phloem channels lie within the 10-100m diameter, which has been shown to be optimal diameter for myoblast alignment through contact guidance. Following 10 days in proliferation media, F-actin filaments were observed to be aligned parallel to the longitudinal axis of the vascular bundle. Subsequently, following 5 days in differentiation media, myoblast maintained an aligned morphology, which led to the formation of aligned myotubes. We therefore conclude that the microtopography of the vascular bundle guides muscle cell alignment. The results presented here highlight the potential of this plant-derived scaffold for in vitro studies of muscle myogenesis, where structural anisotropy is required to more closely resemble in vivo conditions.

The interplay between the extracellular matrix (ECM) mechanical properties and the tumor microenvironment is increasingly recognized as a critical factor in cancer progression. Three-dimensional (3D) culture systems have emerged as essential platforms for in-vitro cell-based applications, offering microenvironments that are more physiologically relevant compared to traditional two-dimensional (2D) cultures. However, independently controlling the topological and mechanical features of 3D matrices remains challenging due to the interdependence of these parameters. In this study, we demonstrate a method for independently tuning pore size and stiffness in collagen I (Coll I) networks and examine their effects on breast cancer and epithelial cell morphology and cluster formation. Collagen concentration was used to modulate bulk stiffness, while polymerization temperature was adjusted to control pore size. Using this approach, we developed a 3D Coll I matrix with tuned stiffnesses from 80, 228 and 360 Pa while simultaneously holding pore size constant (2.5 {micro}m). Similarly, we developed a low- (1.5 mg/mL) and high- (3.5 mg/mL) concentration collagen hydrogel with varying pore sizes from 2.5 {micro}m to 3.1 {micro}m and 2.0 {micro}m to 2.4 {micro}m, respectively, without altering stiffness (80 Pa and 350 Pa). Integrating a breast epithelial cell line, MCF-10A, and metastatic breast cancer cell line, MDA-MB-231, we demonstrate matrix stiffness and pore size independently and differentially regulate cell morphology and cluster formation. Our results establish a robust method for decoupling stiffness and pore size in Coll I matrices enabling more precise investigations into how ECM mechanical properties influence metastatic and epithelial cell behavior. Statement of SignificanceThis study presents a robust method to independently tune stiffness and pore size in 3D collagen I matrices, overcoming a key challenge in extracellular matrix modeling. By decoupling these parameters through collagen concentration and polymerization temperature, the platform enables more accurate investigation of how ECM mechanical properties influence metastatic and epithelial cell behavior. Our finding reveals that matrix stiffness and pore size independently and differentially regulate cell morphology and cluster formation, demonstrating the distinct cellular responses to specific ECM properties and underscoring the importance of the tumor microenvironment in cancer biology and tissue engineering.

Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remain unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, 3D displacement microscopy, matrix remodeling analysis, and YAP nuclear localization, we show that ligand identity governs adhesion organization, force transmission, and mechanotransduction. RGD-functionalized matrices promote {beta}1-integrin clustering, extensive matrix remodeling, strong YAP activation and upregulation of migration-related genes. In contrast, non-adhesive matrices limit adhesion formation, resulting in weak force transmission and amoeboid-like behavior. HAVDI-functionalized matrices induce cadherin clustering and heterogeneous cellular responses, indicating that a hybrid migration mode arises from adhesion organization rather than a distinct transcriptional program. Together, these findings demonstrate that ligand identity alone is sufficient to program migration mode in a force-responsive 3D matrix and provide a versatile platform to dissect cell-matrix interactions in complex environments. Statement of significanceO_LICell migration in tissues is highly adaptable, yet precise control of migration modes in defined 3D biomaterials remains challenging. C_LIO_LIWe introduce fibrous PIC hydrogels presenting RGD, HAVDI, or no adhesive ligands to bias human stem cells toward mesenchymal-like, hybrid, or amoeboid-like migration states. C_LIO_LIBy linking ligand identity to adhesion organization, matrix remodeling, YAP mechanotransduction, and gene expression, this work provides a minimal platform to dissect and engineer 3D cell-matrix interactions C_LI

This study presents the development and characterization of a novel thermosensitive injectable hydrogel designed to enhance the biomechanical properties of poloxamer 407 (P407) through the incorporation of a self-assembling peptide. The primary objective was to engineer a formulation that rapidly gels following intra-articular (i.a.) injection, exhibits improved mechanical strength, and enables sustained release of embedded therapeutic cargo. Gelation time assays demonstrated that the P407-peptide formulation solidified more quickly than P407 alone at equivalent concentrations. Rheological analysis revealed a 1.5 kPa increase in storage modulus in the hybrid hydrogel, confirming improved mechanical integrity. In vitro biocompatibility was assessed using human chondrocytes, with MTS assays and LIVE/DEAD staining indicating no cytotoxicity across tested concentrations. To evaluate in vivo applicability, a near-infrared fluorescent (NIRF) dye was incorporated into the hydrogel and injected intra-articularly into an osteoarthritis (OA) mouse model. The labeled formulation allowed for successful tracking and demonstrated localized gelation, supporting its suitability for site-specific, sustained delivery. Overall, the P407-peptide hydrogel offers a promising platform for i.a. therapeutic applications, combining injectability, rapid thermoresponsive gelation, mechanical reinforcement, and controlled release behavior, making it well-suited for regenerative medicine and OA treatment.

Nanomaterials have been proposed as drug delivery vehicles to enhance targeting and efficiency of traditional and novel therapeutics and have subsequently been studied for potential ecotoxicity. Previous studies have identified size, surface charge, and volume exclusion as factors that influence nanomaterial diffusion and retention. However, there is little accepted or successful quantification of how these parameters influence nanomaterial penetration relative to biological adaptation and biological response. Part of the challenge is the response of living biological interfaces to many of these nanomaterial delivery vehicles and nanosized drugs. This study aimed to emulate key physicochemical barriers to diffusion found in living biomaterials by developing a tunable, synthetic hydrogel. Through the controlled exposure of 150 kDa and 2 MDa nanodextrans with neutral and negative surface charge, we evaluated the systems ability to emulate three core physicochemical features often implicated in biofilm-associated transport resistance: size exclusion, charge interactions, and volume exclusion. We demonstrated a 30% statistically significant decrease in partition coefficients for 2 MDa nanodextran from 150 kDa nanodextran, confirming the ability of the nanocellulose-based microcaps to mimic the permeability of hydrated biomaterial matrices. These findings reflect patterns observed in, for example, living biofilm studies, where size-based diffusion hinderance is commonly reported, but charge-based interaction and volume exclusion are more context-dependent. This controllable system can be coupled with in silico modeling to understand interfacial transport phenomena for nanomaterial-biomaterial interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/703274v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@13c1a34org.highwire.dtl.DTLVardef@dc6c5borg.highwire.dtl.DTLVardef@14dcbd4org.highwire.dtl.DTLVardef@80f70c_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionBoth developing and developed nations have made the creation of innovative wound-healing nanomaterials based on natural extracts a top research goal. The objective of this research was to create a gel containing collagen nanoparticles and evaluate its therapeutic potential for skin lesions. MethodsCollagen nanoparticles from fish scales were produced for the first time using desolvation techniques. Using Fourier transform infrared spectroscopy (FTIR), the structure of the isolated collagen and its similarities to collagen type 1 were identified. The surface morphology of the isolated collagen and its reformulation into nanoparticles were examined using transmission and scanning electron microscopy. Human skin fibroblast cells were employed to examine the cytotoxicity of the nanomaterials, and an experimental model was used to evaluate the wound healing capability. ResultsCollagen nanoparticles formulation was confirmed using FTIR, SEM and TEM analysis. Cytotoxicity studies demomstrated that the manufactured nanoparticles have minor toxicity at high concentrations on human skin fibroblast. Histological investigation proved that the fabricated fish scale collagen nanoparticles promoted the healing process in comparison to the saline group. ConclusionThe fabricated product is a highly influential wound healing product that can be applicable for commercial use. The nanoscale size of collagen nanoparticles, make them interesting candidates for biological applications. Key Summary PointsO_LIThe goal of this research was to create natural, effective wound remedies that could lower health-care costs while also providing pain relief and, ultimately, effective scar repair. C_LIO_LICollagen nanoparticles can be synthesized from fish scale utilizing various nanotechnology-based approaches to stimulate skin cell proliferation and promote wound healing. C_LIO_LICollagen nanoparticles have a rough surface, have a negative potential, and can be used for drug delivery and wound healing. C_LIO_LIHistological and macroscopical analysis showed that the synthesized nanoparticles promoted faster wound healing. C_LI

Three-dimensional (3D) cell cultures, such as spheroids, are increasingly used to perform advanced studies on bone matrix mineralization. However, their full characterization remains challenging. Traditional colorimetric and fluorescent assays using dyes, such as Alizarin Red S (ARS) and calcein, are effective in monolayer cell cultures, but fail to provide reliable information when used in complex 3D cell constructs. In this study, we investigated the application of Coherent Raman Scattering microscopy for label-free, comprehensive characterization of extracellular matrix (ECM) mineralization in alginate-encapsulated bone spheroids. After confirming that traditional staining techniques are unreliable for mineral detection in spheroids, Stimulated Raman Scattering (SRS) microscopy was used to detect phosphate-rich mineral deposits at a Raman shift of 960 cm-1, while Second Harmonic Generation (SHG) microscopy was used in association with SRS to provide complementary information on the deposition and organization of the collagenous matrix. SRS was used to detect lipid-rich regions at a Raman shift of 2857 cm-1 to perform cell localization. SRS imaging revealed the presence of phosphate-rich regions in the spheroids, including the core regions, usually challenging to characterize in intact 3D constructs. Raman spectral scans on SRS-positive regions confirmed the specificity of the phosphate signal. In addition, comparison of SRS and Coherent Anti-Stokes Raman Scattering (CARS) demonstrated the advantage of SRS in terms of reduced background compared to CARS for lipid imaging. Taken together, our results demonstrated that SRS, in combination with SHG, provides a promising and powerful approach to perform label-free, chemically specific characterization of intact 3D bone models.

Efforts to model and repair connective tissue through engineered tissue constructs have generated great interest in culturing cells in 3d polymer network environments. It has been shown that the polymer environment is influential in determining cellular responses such as differentiation, migration and morphology. Hydrogels are used to mimic the cellular microenvironment, but in most cases hydrogels consisting of one polymeric component are used whereas tissues are composites of different polymers. A clear understanding of how different extracellular components and their mechanical characteristics influence cell behaviour is lacking. Here we developed and characterised composite hydrogels of hyaluronan and fibrin and evaluated their use for cartilage tissue engineering. We demonstrate that these cartilage-mimicking composites have a higher stiffness relative to the individual constituents. Next, we cultured human mesenchymal stromal cells in these 3D hydrogels with chondrogenic media and revealed marked differences in cell morphology, gene expression and cartilage-like matrix deposition depending on the specific extracellular composition. We found that, despite evidence for strong adhesion of the cells to fibrin networks in 2D systems, in 3D systems the primary determinant of cellular morphology is the significantly denser hyaluronan network. Dense hyaluronan hydrogels cause local cell confinement evidenced by rounder cell morphologies, independent of the presence of fibrin. While the composite fibrin-hyaluronan hydrogels led to lower expression of chondrogenic genes than hyaluronan alone, the larger linear modulus and resistance to cell-mediated contraction due to the composite nature of the matrix provides a strong advantage in terms of macroscopic mechanical stability. These findings highlight the potential of multi-component hydrogels for controlling cellular behaviour and bulk mechanical properties of cell-hydrogel constructs independently, therefore opening avenues for better understanding the complex interplay between cells and their extracellular environment and thus improve the biofabrication of connective tissues for disease modelling and tissue regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/555478v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151962org.highwire.dtl.DTLVardef@1358890org.highwire.dtl.DTLVardef@198dcedorg.highwire.dtl.DTLVardef@d04f80_HPS_FORMAT_FIGEXP M_FIG C_FIG

The severe, long-lasting harm caused by plastic pollution to marine ecosystems and coastal economies has led to the development of biodegradable plastics; however, their limited decomposition in cold, dark marine environments remains a challenge. Here, we present our newly developed technologies for creating 3D-bioprinted living materials for bioplastic degradation with specific use in marine environments. Our approach integrates halotolerant bioplastic-degrading bacterium Bacillus sp. NRRL B-14911 into alginate-based bio-ink to print an engineered living material (ELM) termed a "bio-sticker." Quantification of bacteria viability reveals that bioprinted marine bacteria survive within bio-stickers for more than three weeks. The rate at which the bio-stickers degrade the bioplastic polyhydroxybutyrate (PHB) can be tuned by altering bio-sticker biomass concentration, bioplastic concentration, or incubation temperature. Bio-stickers that are transferred to a new PHB sample still retain high biodegradation activity, demonstrating their durability. Strain sweep oscillatory tests demonstrate viscoelastic behavior of the bio-stickers. Monotonic tensile tests indicate that the elastic modulus and the adhesion of the bio-stickers are not negatively impacted by bacteria growth or incubation temperature. Our work paves the way for development of ELMs to facilitate the inclusion of bioplastics within the blue economy, promoting the emergence of more sustainable and eco-friendly materials.

We introduce a platform for the fabrication of customizable wound healing dressing. The platform integrates electrospun nanofibers, bioprinted hydrogels, and cellular spheroids into hierarchical, fiber-reinforced hybrid constructs. The construct leverages the mechanical strength of polycaprolactone (PCL) nanofibers and the ECM-like properties of GelMA/PEGDA hydrogel. These materials support the incorporation of bone marrow-derived mesenchymal stem cell (BM-hMSC) spheroids, which act as a supportive "cell niche," enhancing the viability of the hMSC during and after bioprinting, and facilitating their spreading across the construct during the maturation phase. The characterization of the hybrid constructs demonstrated strong structural integrity and enhanced mechanical properties, making them well-suited for clinical wound dressing applications. In vitro assays, including live/dead staining, MTT assays, and scratch assays, revealed increased cell attachment, proliferation, and migration. The spheroids maintained their viability over extended periods, significantly contributing to wound closure in the scratch assay. This innovative approach, which combines electrospinning and light-based bioprinting, offers a promising strategy for the development of customizable wound dressings that closely adapt to the complex architecture of human skin. The bioprinting approach allows for the creation of tailored geometries for specific clinical requirements. Future research will focus on optimizing scaffold design and conducting long-term in vivo studies to validate the platforms clinical potential.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material, but also by ECM geometry and cell density. We hypothesised that these factors would influence cell-traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix-deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared to soft hydrogels, or on thin compared to thick hydrogels. Cell-imparted matrix deformations were greater on soft compared to stiff hydrogels or thick compared to thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

In native extracellular matrices (ECM), cells can use matrix metalloproteinases (MMPs) to degrade and remodel their surroundings. Likewise, synthetic matrices have been engineered to facilitate MMP-mediated cleavage that enables cell spreading, migration, and interactions. However, the intersection of matrix degradability and mechanical properties has not been fully considered. We hypothesized that immediate mechanical changes result from the action of MMPs on the ECM and that these changes are sensed by cells. Using atomic force microscopy (AFM) to measure cell-scale mechanical properties, we find that both fibrillar collagen and synthetic degradable matrices exhibit enhanced stress relaxation after MMP exposure. Cells respond to these relaxation differences by altering their spreading and focal adhesions. We demonstrate that stress relaxation can be tuned through the rational design of matrix degradability. These findings establish a fundamental link between matrix degradability and stress relaxation, which may impact a range of biological applications. Table of contentsThis work reveals that matrix degradability, through its effects on stress relaxation, is an important cellular mechanotransduction cue. Cell-scale mechanical characterization shows that collagen gels and degradable synthetic gels display enhanced stress relaxation post-degradation. Stress relaxation is then tuned by systematically varying degradability, resulting in the regulation of cell spreading. This identifies degradability as a key chemomechanical design feature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/605514v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13a701dorg.highwire.dtl.DTLVardef@196bb05org.highwire.dtl.DTLVardef@d04613org.highwire.dtl.DTLVardef@1a8b1d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

The organization of epithelial tissues with precise spatial definition is essential to various biological processes and to generate curved epithelial structures. However, the regulation of the architecture and dynamics of collective epithelial assemblies by the matrix curvature remains understudied. Here, we photopolymerize microwells of various diameters in hydrogels to form curved epithelial structures such as breast epithelial lobules, and study how in-plane and out-of-plane curvatures modulate the mechanoresponse of epithelial tissues. In-plane curvature governed by the microwell radius drives the centripetal orientation of cells and nuclei close to the edge of the microwell, resulting from contractile forces exerted by a supracellular actomyosin purse-string. Convex out-of-plane curvature imposed at the microwell entrance leads to a vertical orientation of the nuclei towards the microwell axis. We demonstrated that increasing the out-of-plane curvature leads to more flatten and elongated nuclear morphologies with high levels of compacted chromatin. Epithelial cells exhibit higher directionality and speed around the microwell edge, demonstrating that the out-of-plane curvature significantly enhances the cellular trafficking. These findings demonstrate the importance of in-plane and out-of-plane curvatures in epithelial organization and how both can be leveraged to facilitate the engineering of curved structures to study curvature-dependent mechanotransduction pathways.

The combination of synthetic biology and additive manufacturing has driven major changes in production of biomaterials, especially through the use of three-dimensional (3D) bioprinting to create engineered living materials. However, current fabrication methods can be limited by prohibitive hardware costs and the inability to maintain structural fidelity in complex, free-form living architectures. This work demonstrates how to build a low-cost, open-source 3D bioprinting platform that can make complicated bacterial structures with complex geometry and high dimensional accuracy. A commercially available, conventional fused deposition modeling 3D printer was modified to create a bioprinting system that is simple to build. The modified bioprinter, which costs around $450, is less expensive than many commercial bioprinters. This 3D-printing technology uses slurry-based support bath methods featuring low-cost gelatin and agarose microparticles, resulting in structures with a high aspect ratio (>8:1) and feature sizes as small as 260 m. The optimization of critical printing settings, including the ability of the bioink to retract during non-print movements, resulted in a reduction of unwanted bacterial deposition by nearly two orders of magnitude. Long-term viability experiments showed that bacteria in the bioprints could survive for at least 28 days with nutrient supplementation. Additionally, 3D-printed engineered biofilms revealed that incubation conditions and extracellular matrix composition significantly impacted the mechanical properties of printed constructs, with tradeoffs between matrix production and mechanical integrity. This study showcases an accessible 3D bioprinting platform for advanced bioprinting technologies, enabling development of engineered living materials with potential applications in synthetic biology, biotechnology, and tissue engineering.

The dynamic, viscoelastic nature of the extracellular matrix (ECM), with variations in stiffness and viscosity, plays a key role in cellular behaviors like mechano-sensing, migration, and force generation. Viscoelastic hydrogels are important for mimicking the ECM to study cell migration and tissue engineering. However, creating hydrogels with continuous viscoelastic gradients is challenging due to issues with spatial resolution, reproducibility, and accurately replicating native tissue properties. Here we present a novel approach to generate hydrogels with precisely controllable viscoelastic gradients using drop-by-drop condensation. This approach allows fine-tuned replication of the tissue-specific local mechanical properties, resulting in hydrogels of heterogeneous viscoelasticity. Directed migration and separation of multiple cell lines are thus driven by local viscosity and elasticity rather than solely stiffness. By incorporating non-linear mechanical gradients, this technique provides insights into ECM viscoelasticitys role in cellular behavior and offers a versatile platform for advanced tissue engineering and regenerative medicine applications.

The study of in vitro models of breast cancer is crucial for understanding and treating the malignancy in patients, with 3D in vitro models providing researchers with more biomimetic systems to overcome limitations of current to 2D cultures and in vivo animal models. Ex vivo patient tissues have shown that malignant breast tissues are stiffer than healthy or benign tissues, and that the stiffness corresponds with increasing tumour grade. Stiffening of the breast tumour environment alters tumour cell phenotype and facilitates tumour progression, invasion and metastasis. Better understanding of the relationship between extracellular matrix stiffness and breast cancer cell phenotype, and how that is important in the initiation of metastasis, should lead to designing 3D models that mimic the breast tumour microenvironment at different stages of breast cancer progression. This study investigated phenotypic response of two breast cancer cell lines that are representative of clinical breast cancer subtypes (MCF7, Luminal A; MDA-MB-231, Triple Negative Breast Cancer) in gelatin-methacryloyl (GelMA) hydrogels of varying stiffness. A visible light photoinitiation system was adopted to provide a tuneable photocrosslinking platform to systematically control hydrogel stiffness and tumour microenvironment. This allowed rapid fabrication of biocompatible hydrogels supporting high cell viability over long-term culture. The impact of a clinically relevant range of microenvironmental stiffness on breast cancer cell behaviour and phenotype was examined over a 21-day culture period using GelMA hydrogels. Results showed that MCF7 cells cultured for 21 days in high stiffness hydrogels (10 wt%; 28 kPa) responded by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers N-cadherin and Vimentin, whereas MDA-MB-231 cells showed no changes in EMT-markers when cultured in hydrogels of corresponding stiffness (10 wt%; 33 kPa). Culturing both cell lines in soft hydrogels (5 wt%; 11 kPa) maintained their phenotype over 21 days, highlighting the importance of controlling hydrogel mechanical properties when studying breast cancer cell phenotype.

Mucointegration is as important as osseointegration to ensure the survival of implant-supported prosthesis. Indeed, effective soft tissue integration (STI) prevents the appearance of complication through bacterial dissemination. To optimize STI, electrochemical anodization can be used to nanostructure the trans-gingival part of the prosthetic component. Moreover, Selective Laser Melting (SLM) is a new 3D-manufacturing technique that enables the production of customized implant-supported prosthesis with complex geometry. ObjectiveThe aim of this study is to evaluate the effect of a SLM manufactured and anodized Ti6Al4V surface on the behaviour of both, human gingival fibroblasts and oral bacteria. MethodSLM-Ti6Al4V discs were polished and anodized with defined parameters to obtain nanotubes (NTs) with specific morphology. Surface characterization was assessed through surface topography and wettability. Human gingival Fibroblasts were cultured, and cell morphology was observed by SEM at day 7. Proliferation, viability (day 1,4,7) and adhesion (6 h and 36 h) were analyzed. Then immunofluorescence and RT-qPCR were used to detect the distribution and the gene expression of vinculin at 48 h. An early colonizer (Streptococcus gordonii) was used for a parallel evaluation of bacteriological adhesion. ResultsSLM-ANO-Ti6Al4V showed similar performances in terms of cytotoxicity, compared with a machined and polished titanium surface currently used in clinics. Interestingly, cell adhesion was enhanced on anodized SLM surfaces, with a difference in the distribution of focal adhesion plaques in HGFs, while biofilm formation of S. gordonii was not affected by anodization. SignificanceSLM anodized surface showed promising ability to promote STI while controlling bacterial adhesion.

Diabetic kidney disease (DKD) is often diagnosed only after irreversible damage, limiting early treatment. The kidney mesangium, a structure highly sensitive to mechanical forces, plays a key role in early fibrosis development, yet its response to early disease cues like stiffness and biochemical changes is poorly understood. This is due to limitations in current in vitro models, poor in vivo accessibility, and static biopsy samples. To address this, we developed a 3D in vitro model of the mesangial microenvironment using stiffness-tunable gelatin methacrylate (GelMA) hydrogels that mimic healthy and fibrotic kidney tissue. Exposing mesangial cells to glucose and TGF-{beta}1 led to altered cell shape, increased dry mass, and elevated expression of fibrotic markers (-SMA and collagen IV), especially under stiffer conditions, indicating a synergistic effect of biochemical and mechanical stress. These responses were integrated using Gaussian process regression to create a 3D "severity cube" that maps DKD progression across mechanical and chemical inputs. This system quantifies early mesangial transitions and reveals key fibrosis-related mechanisms. By combining organotypic modeling with interpretable inference, our platform offers a predictive tool for early disease stratification and a basis for studying subclinical fibrosis progression in DKD.

Intravasation is the process by which cancer cells breach the physical boundaries of a primary tumor and enter blood or lymphatic vessels. In this work, MCF-7 breast cancer cells were cultured within polymer-based microcapsules (here referred to as artificial microtumors) to investigate the transcriptomic and morpho-mechanical changes occurring in cancer cells during their release from these matrices, mimicking in vitro the process of intravasation. Our results show that even confined and released cancer cells share approximately 95% of their global transcriptomic profiles, intravasation-like cells exhibited marked differences in the expression of pathogenic hallmarks, including pathways associated with cell proliferation, immunosurveillance, and dormancy. Notably, a clear upregulation of YAP/TAZ signaling was observed in released cells, a result further supported by single-cell traction force microscopy assays, demonstrating that those cells exhibit higher biomechanical activity compared to cells located within artificial microtumors or those cultured on conventional 2D flasks, as shown for intravasated cells in vivo. To further enrich our investigation, the mechanotranscriptomic activity of MCF-7 cells was compared with suspended spheroids cultured on non-adherent surfaces (i.e., agarose hydrogels). Our results show that released cells displayed increased biomechanical activity and elevated expression of malignant markers, indicating that mechanical stress, beyond cell-cell contact alone, is required to trigger malignant responses. These observations were further supported by co-culture studies of MCF-7 cells with human fibroblasts and endothelial cells, which showed reduced proliferative and invasive capacities under confinement. Overall, our findings demonstrate that shifts in mechanical and metabolic stress, as experienced during intravasation, act as critical stimuli driving mechanotranscriptomic programs associated with cancer progression.

Extracellular matrix (ECM) proteins assemble to form a heterogeneous connective scaffold that supports cells. Physical interactions between cells and the matrix regulate cellular behaviors and influence subsequent tissue construction. However, there is a lack of fundamental understanding regarding the contributions of individual native ECM proteins to the matrix. This gap arises from the need for nanoscopic characterization, which operates on a much smaller length scale than typical assessments in cell and tissue cultures, as well as in tissue reconstruction and clinical implantation. This study aims to systematically investigate how individual ECM proteins affect lipid membranes structurally and mechanically, and how these influences regulate cell migration. Results from Langmuir isotherm analysis, X-ray reflectivity measurements, and cell scratch assays demonstrate that strong collagen adsorption on the membrane surface disrupts lipid packing. However, its rigid network provides a sturdy scaffold for cell adhesion, thereby enhancing cell attachment and promoting cell migration. In contrast, elastin has a minimal structural or mechanical impact on the membrane during both adsorption and compression, but it benefits cells by facilitating migration and reducing the risk of infection. Fibronectin, on the other hand, exhibits complex mechanical responses to compression, characterized by significant structural rearrangements that occur during adsorption. This strong interaction with the membrane can result in excessively high adhesion forces, ultimately limiting cell motility. These findings lay the foundation for the design of artificial scaffolds that can manipulate cellular responses, a critical step toward advancing regenerative medicine and tissue engineering. SignificanceFabricating extracellular matrix (ECM) scaffolds from cells offers advantages over traditional approaches, such as decellularized tissues, which face donor limitations, and artificial scaffolds, which may hinder cellular communication. However, the slow harvesting process of cell-derived ECM has limited its clinical applications. This research is part of a larger mission to engineer ECM prescaffolds on lipid carriers tailored to cell requirements, enhancing ECM production and regulating cell behavior. The first step involves systematically analyzing the structural and mechanical effects of ECM on lipid membranes and how these effects regulate cellular behavior. This work confirms distinct characteristics of ECM proteins, advancing fundamental understanding of cell-matrix interactions and paving the way for scaffold engineering.