Advanced Materials Interfaces

The catch bond complex between serine-aspartate repeat protein G (SdrG) from Staphylococcus epidermidis and the beta chain of fibrinogen (Fg{beta}) exhibits two distinct rupture populations when dissociated under tensile force. Such complexes present exciting possibilities for developing dynamic biomaterials due to their unique response to shear force. However, the environmental responsiveness of this complex and its influence on adhesion behaviour in multi-valent systems remain underexplored. Using AFM-single molecule force spectroscopy (AFM-SMFS) and spinning disk adhesion (SDA) assays, we examined how protein orientation, mutations, and environment influence the stability and scaling behaviour of this catch bond system. Our findings confirmed that anchor point location (i.e., the direction from which the protein is pulled) strongly influences catch bond behaviour, while an S338H mutation in the binding domain destabilised the interaction in both single-molecule and multi-valent adhesion assays. This research examines how catch bond behaviour translates from the nanoscale to microscale using single molecule and multi-valent cell adhesion measurements and provides a toolkit for exploiting catch bonds towards macroscale material applications.

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features puncture adhering cells, thus also reducing MSC adhesion. Here, we use a poly(ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. This nanotopography when combined with the PEA coating enhanced osteogenesis and reduced adhesion of Pseudomonas aeruginosa in culture. Using a novel MSC-Pseudomonas aeruginosa co-culture, we also show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection and supporting osteogenic repair.

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by: 1) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell amplification, 2) donor-specific characteristics including MSC frequency/quality that decline with disease state and increasing age, 3) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which pose safety and consistency issues. To overcome those limitations while enabling robust MSC production with constant high yield and quality, we developed a chemically defined biomimetic surface coating, called isoMATRIX, that facilitates the isolation of significantly higher numbers of MSCs in xeno-/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. In sum, the isoMATRIX promotes enhanced xeno-, serum-free, or chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Bacterial biofilms are highly adaptable and resilient to challenges. Nutrient availability can induce changes in biofilm growth, biomass, morphology, architecture and mechanical properties. Bacterial extracellular matrix plays a major role in achieving biofilm stability under different environmental conditions. Curli amyloid fibers are determining for the architecture and stiffness of E. coli biofilms, but how this major matrix component adapts to different environmental cues remains unclear. Here, we investigated for the first time the effect of nutrient availability on both i) biofilm materials properties and ii) the structure and properties of curli amyloid fibers extracted from the biofilms. For this, we cultured E. coli W3110, which main matrix component is curli fibers. We quantified the size, mass and water content of the resulting biofilms and estimated their mechanical properties by microindentation. The curli amyloid fibers were then purified from the biofilms and their molecular structure and properties were studied by spectroscopic techniques. Our results show that the availability of nutrients in the substrate influences the yield of curli fibers, their structural composition and chemical stability, and suggest that these molecular features contribute to the stiffness of the biofilms. Biofilms grown on substrates with high nutrient concentration are softer, contain less curli fibers, and these fibers exhibit low {beta}-sheet content and chemical stability. Our multiscale study sheds new light on the relationship between the molecular structure of bacterial matrix and the macroscopic properties of biofilms. This knowledge will benefit the development of both anti-biofilm strategies and biofilm-based materials. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/556686v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@ab9469org.highwire.dtl.DTLVardef@9c8f61org.highwire.dtl.DTLVardef@895de8org.highwire.dtl.DTLVardef@74433d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

E. coli biofilms consist of bacteria embedded in a self-produced matrix mainly made of protein fibers and polysaccharides. The curli amyloid fibers found in the biofilm matrix are promising versatile building blocks to design sustainable bio-sourced materials. To exploit this potential, it is crucial to understand i) how environmental cues during biofilm growth influence the molecular structure of these amyloid fibers, and ii) how this translates at higher length scales. To explore these questions, we studied the effect of water availability during biofilm growth on the conformation and functions of curli. We used microscopy and spectroscopy to characterize the amyloid fibers purified from biofilms grown on nutritive substrates with different water contents, and micro-indentation to measure the rigidity of the respective biofilms. The purified curli amyloid fibers present differences in the yield, structure and functional properties upon biofilm growth conditions. Fiber packing and {beta}-sheets content correlate with their hydrophobicity and chemical stability, and with the rigidity of the biofilms. Our study highlights how E. coli biofilm growth conditions impact curli structure and functions contributing to macroscopic materials properties. These fundamental findings infer an alternative strategy to tune curli structure, which will ultimately benefit to engineer hierarchical and functional curli-based materials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/517345v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@124ddd2org.highwire.dtl.DTLVardef@15f234dorg.highwire.dtl.DTLVardef@106a65forg.highwire.dtl.DTLVardef@194c53f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Copper is involved in the biosynthesis of collagen, however soluble copper salts dissipate quickly and copper nanoparticles are cytotoxic. Here we added a novel copper-containing nanomaterial (CuHARS) to assess human chondrocyte function in the presence of copper. Human dermal fibroblasts (HDFs) were also treated as a control. Chondrocyte response to CuHARS was assessed by chronic nanomaterial treatment (30 {micro}g/ml) followed by digital microscopy and image analysis of cellular features compared to normal chondrocytes. Unexpectedly, chronic CuHARS treatment of human chondrocytes transformed cells over time to cells with extremely elongated and variegated processes and lower proliferation rates compared to normal chondrocytes. In these transformed cells, which we named 3G, shedding of fine processes was observed over time and collected supernatants demonstrated elevated collagen levels compared to normal cell culture media. In contrast to chondrocytes, HDFs treated with CuHARS showed attenuated changes in morphology, and notably retained a prominent ability for continued proliferation. These results demonstrate that a copper-containing biohybrid material (CuHARS) can stably transform human chondrocytes with highly altered morphology, lower proliferation rates, and altered membrane dynamics compared to normal chondrocytes. In contrast, human dermal fibroblasts demonstrated attenuated changes in morphology, and retained an enhanced ability for proliferation.

Long bone fractures are primarily repaired through endochondral ossification, a process in which a soft cartilage template forms at the injury site and is gradually replaced by bone. While bone has an innate self-healing capacity, this process can be disrupted in cases of large or complex defects, where regeneration fails, and clinical intervention is required. This study aimed at the development of a tissue engineering approach using human periosteum-derived cell (hPDC) spheroids encapsulated or bioprinted at high density within hyaluronic acid methacrylate (HAMA) hydrogels to support hypertrophic cartilage formation as a template for endochondral bone regeneration. We first compared different encapsulation time points (days 1, 7, and 14), finding that early encapsulation (day 1) enhanced spheroid fusion, increased DNA content, and promoted hypertrophic cartilage formation, as indicated by greater glycosaminoglycan (GAG) and collagen deposition along with lacunae formation. Next, HAMA-encapsulated spheroids were compared to spheroids formed using a standardized microwell platform, demonstrating that encapsulation promoted a more mature cartilage-like matrix with thicker collagen fibers and enhanced hypertrophic differentiation. Gene expression and immunostaining confirmed progression toward hypertrophic and osteogenic phenotypes. Finally, extrusion-based bioprinting of HAMA bioinks comprising a high-density of hPDC spheroids demonstrated scalability, improved spheroid alignment, and maintained robust cell viability and hypertrophic differentiation. HAs bioactivity and regulatory advantages support clinical translation, although achieving spatial control remains an area for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/674866v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@40b55eorg.highwire.dtl.DTLVardef@434b2corg.highwire.dtl.DTLVardef@1fc4640org.highwire.dtl.DTLVardef@168252e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fetuin-A is a plasma protein of interest for bone-interfacing applications due to its role in mineralization processes through calcium/phosphate ion-binding capabilities. However, the role of fetuin-A in the initial stages of cellular interaction with biomaterials and the mechanisms involved are not fully clear. This work investigated the response of osteoblast-like Saos-2 cells to model gold substrates presenting pre-adsorbed fetuin-A as a surface modification, to determine the role of the protein in cell attachment and proliferation. Correlative quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance, and radiolabeling confirmed fetuin-A adsorbed on model surfaces in similar quantities compared to serum albumin but formed a less packed layer with increased water entrapment. Surfaces presenting pre-adsorbed fetuin-A enhanced cellular adhesion, similar to fibronectin, but attached cells displayed morphological characteristics more similar to those with pre-adsorbed albumin, with lower average surface area and maximum axis. Over 3 days, fetuin-A exhibited lower cellular proliferation compared to the fibronectin control, likely correlated to the decrease in cellular metabolism observed at the same time-point, and persisted over 7 days. These results provide insight into the role of adsorbed fetuin-A for bone-interfacing implant applications, suggesting the pre-adsorption of the protein alone aids cellular attachment, but is not sufficient to promote early stages of osseointegration.

Droplet impacts on various surfaces play a profound role in different bio-physiological processes and engineering applications. The current study opens a new realm that investigates the plausible effect of impact velocities on bacteria-laden droplets against a solid surface. We unveiled the alarming consequences of Salmonella Typhimurium (STM) laden drop, carrying out the in vitro and intracellular viability of STM to the impact Weber numbers ranging from 100-750. The specified Weber number range mimics the velocity range occurring during the respiratory processes, especially the airborne dispersion of drops during cough. A thick ring of bacterial deposition was observed in all cases irrespective of impacting velocity and the nutrient content of the bacterial medium. The mechanical properties of the bacterial deposit examined using Atomic Force Microscopy reveals the deformation of bacterial morphology, cushioning effect and adhesion energy to determine the cell-cell interactions. The impact velocity induces the shear stress onto the cell walls of STM, thereby deteriorating the in vitro viability. However, we found that even with compromised in vitro viability, Salmonella retrieved from deposited patterns impacted at higher velocity revealed an increased expression of phoP (the response regulator of the PhopQ two-component system) and uninterrupted intracellular proliferation in macrophages. The inability of STM{Delta} phoP growth in nutrient-rich dried droplets to the subjected impact velocities signifies the predominant role of phoP in maintaining the virulence of Salmonella during desiccation stress. Our findings open a promising avenue for understating the effect of bacteria-laden drop impact and its role in disease spread. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Thrombogenicity causes significant complications in the application of blood-contacting implants, requiring strategies to prevent adverse coagulation reactions. The thrombotic responses to the foreign surfaces are mainly driven by surficial factors such as surface energy, topography, and electrochemical interactions. Although anticoagulation therapies reduce the risks of clotting, patients might still encounter bleeding complications. Therefore, rather than high-risk anticoagulation therapies to counteract coagulation, it is essential to ensure hemocompatibility through the materials intrinsic properties. Endothelialization is crucial in preventing thrombotic complications, with various strategies explored for facilitating endothelial cell adhesion and proliferation. We investigated the impact of crystallographic anisotropy on endothelial and blood cell interactions on four main planes (A-, C-, M-, and R-planes) of single crystalline alumina (-Al2O3, sapphire). Employing advanced surface characterization techniques, including SIMS, KPFM and Zeta potential measurements, our study sheds light on the hemocompatibility of biomaterials considering anisotropic effects. We elucidated that the A-plane of alumina promotes endothelialization and suppresses platelet activation in contrast to other crystallographic planes. Our investigation into cell-surface interactions provides valuable insights and contributes to the advanced biomaterial design, ultimately leading to enhanced clinical outcomes.

The global decline of coral reefs calls for new strategies to rapidly restock coral populations and maintain ecosystem functions and services. Low recruitment success on degraded reefs hampers coral sexual propagation and contributes to limited genetic diversity and reef resilience. Here, we introduce a living bacteria-powered reef ink (Brink) for assisted coral recruitment. Brink can be rapidly applied to restoration substrates via photopolymerization, and it has been formulated to cultivate two settlement-inducing bacterial strains (Cellulophaga lytica and Thalassotalea euphylliae). Settlement assays performed with broadcast spawning (Montipora capitata) and brooding (Pocillopora acuta) Indo-Pacific corals showed that Brink-coated substrates increased settlement >5-fold compared to uncoated control substrates. Brink can be applied as a coating or 3D bioprinted, leading to various potential applications for integration with reef engineering. Our approach underscores the potential of using functional living materials for augmented ecosystem engineering and reef rehabilitation. SynopsisThis study introduces a functional and sustainable bacteria-powered living material that enhances coral settlement, promoting coral reef rehabilitation and ecosystem resilience.

Cells attach to the world around them in two ways--cell:extracellular-matrix adhesion and cell:cell adhesion--and conventional biomaterials are made to resemble the matrix to encourage integrin-based cell adhesion. However, interest is growing for cell-mimetic interfaces that mimic cell-cell interactions using cadherin proteins, as this offers a new way to program cell behavior and design synthetic implants and objects that can integrate directly into living tissues. Here, we explore how these cadherin-based materials affect collective cell behaviors, focusing specifically on collective migration and cell cycle regulation in cm-scale epithelia. We built culture substrates where half of the culture area was functionalized with matrix proteins and the contiguous half was functionalized with E-cadherin proteins, and we grew large epithelia across this Janus interface. Parts of the tissues in contact with the matrix side of the Janus interface exhibited normal collective dynamics, but an abrupt shift in behaviors happened immediately across the Janus boundary onto the E-cadherin side, where cells formed hybrid E-cadherin junctions with the substrate, migration effectively froze in place, and cell-cycling significantly decreased. E-cadherin materials suppressed long-range mechanical correlations in the tissue and mechanical information reflected off the substrate interface. These effects could not be explained by conventional density, shape index, or contact inhibition explanations. E-cadherin surfaces nearly doubled the length of the G0/G1 phase of the cell cycle, which we ultimately connected to the exclusion of matrix focal adhesions induced by the E-cadherin culture surface.

Wet bonding is a basic technique used daily in clinics for tooth-restoration fixation. However, only 50% of the bonding lasts more than 5 years and thus patients must visit the dentists repeatedly. This is attributed to the limited infiltration of adhesives into the demineralized dentin (DD) matrix during wet-bonding. Herein, we show that reconciling interfacial compatibility conflict between the DD matrix and the critical hydrophobic adhesive molecules via hydrophobizing the DD matrix enables the adhesives to thoroughly infiltrate and uniformly distribute within the DD matrix. Thus, the bonding of the hydrophobic DD matrix using commercial dentin adhesives achieves the bonding strength 2-6 times higher than that of the non-treated DD matrix. When a hydrophobic adhesive is applied on the hydrophobic DD matrix, a flawless hybrid layer is produced as observed by nanoleakage investigation. A long-term bonding strength comes up to 7.3 fold that of the control group and very importantly, with no attenuation after 12 months. This study clarifies the basic cause of poor wet-bonding durability and demonstrates a paradigm in adhesive dentistry to overcome the long-existing bonding durability problem associated with inadequate adhesive infiltration into the DD matrix. This provides a new angle of view to resolve clinical dentin bonding durability problem and will significantly promote adhesive dentistry. HighlightsO_LIInherent interfacial compatibility conflict between demineralized dentin matrix and hydrophobic molecules of dentin adhesives is the basic cause for the dentin bonding durability problem. C_LIO_LIReconciling the interfacial compatibility conflict markedly facilitates adhesive infiltration in the demineralized dentin matrix and greatly enhances bonding effectiveness. C_LIO_LIHigh interfacial compatibility produces a flawless hybrid layer and ideal bonding effectiveness and durability. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/430396v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@9687e1org.highwire.dtl.DTLVardef@b2fc79org.highwire.dtl.DTLVardef@dd7379org.highwire.dtl.DTLVardef@c8117b_HPS_FORMAT_FIGEXP M_FIG For wet bonding, poor infiltration of adhesives within the DD matrix inevitably produces numerous defects throughout the hybrid layer, which always leads to the failure of restoration. Via hydrophobizing the DD matrix, reconciling interfacial compatibility conflict between the DD matrix and the hydrophobic adhesive monomers overcomes durability problems associated with the infiltration of adhesives into the DD matrix producing a flawless hybrid layer and providing ideal bonding effectiveness and durability. C_FIG

The potential of electrical stimulation for cell control in tissue engineering remains largely underestimated, as does the use of organic semiconductors. The tunable physico-chemical properties of organic semiconductors offer advantages over commonly used inert metals. This study investigates the effects of pulsed electrostimulation and electrode materials, gold and poly(3,4-ethylenedioxythiophene) (PEDOT), on the physiological functionality of human vascular endothelial cells. A novel electrostimulation platform incorporating gold or PEDOT electrodes was developed and characterized for electrical performance. Human umbilical vein endothelial cells were cultured on this platform, and the effects of electrostimulation and electrode material were assessed through morphological analysis, nitric oxide (NO) production, and expression of key endothelial marker genes. PEDOT electrodes produced higher electrical current during electrostimulation. Interestingly, cell morphology, including elongation and alignment, showed minimal changes under electrostimulation. NO production, a key marker of vascular health, was enhanced by electrostimulation, with PEDOT electrodes showing a trend toward greater NO accumulation than gold. Gene expression analysis revealed material- and stimulation-specific trends: electrostimulation generally upregulated KLF2, KLF4, and CYP1B1 on PEDOT electrodes but suppressed their expression on gold electrodes. These findings suggest that PEDOT electrodes, with their enhanced electrochemical properties and ability to support endothelial functionality, provide a safe and efficient platform for endothelial cell electrostimulation. This study advances understanding of the interplay between material properties and electrostimulation and highlights PEDOT as a promising candidate for vascular tissue engineering and regenerative medicine.

Blood clotting is the bodys natural reaction in wound healing and is also the cause of many pathologies. Fibrin - the main protein in the clotting process provides clots mechanical strength by forming a scaffold of complex fibrin fibers. Fibrin fibers exhibit high extensibility and primarily elastic properties under static loading, which differ from in vivo dynamic forces. In many biological materials, the mechanical response changes under repeated loading/unloading (cyclic loading). Using lateral force microscopy, we show fibrin fibers possess viscoelastic behavior and experience irreversible damage under cyclic loading. Cross-linking results in a more rigid structure with permanent damage occurring mostly at larger strains, which is corroborated by computational modeling of fibrin extension using a hyperelastic model. Molecular spectroscopy analysis with broadband coherent anti-Stokes Raman scattering spectroscopy in addition to molecular dynamic simulations allow identification of the source of damage, the unfolding pattern, and inter and intramolecular changes in fibrin. The results show partial recovery of proteins secondary and tertiary structures under load, providing deeper understanding of fibrins unique behavior in wound healing or pathologies like stroke and embolism.

Detection of micro- and nanoplastics (MNPs) in human tissues has raised growing concern about their biological effects on tissue and cell function. While previous studies have examined MNP-cell interaction, most focused on limited cell and plastic types. Here, we present a comprehensive, quantitative investigation into how different types of nanoplastics (NPs) associate with and affect diverse cell types under physiologically relevant conditions. Using microfluidic-calibrated fluorescence microscopy, we quantify NP accumulation in cells in vitro and match cellular NP concentrations to levels reported in human tissues. While cell-associated NPs could be gradually released in vitro, they persist in vivo for over one month without detectable reduction in a mouse model. We discover that NP exposure at these levels broadly impairs cell proliferation across epithelial, endothelial, fibroblast, and immune cells, with cell type-dependent sensitivity. NP exposure also reduces motility in T cells and fibroblasts, with more complex effects observed in macrophages. Mechanistically, NP-cell association and trans-epithelial transport involved not only classical endocytic regulators but also pathways related to ion and water transport. Notably, NP association and release were highly sensitive to the extracellular fluid environment within the physiological range. By testing inhibitors of these pathways, we identified molecules that reduce NP-cell association and promote release. We further compared common NPs found in human samples and widely used in research: polystyrene (PS), polyethylene (PE), and polypropylene (PP). Although these NPs similarly impaired proliferation and motility, they showed markedly different cellular association and release dynamics. These findings reveal the impact of NPs on tissue cell functions and uncover novel regulatory pathways, establishing a quantitative framework for studying NP-cell interactions in biologically relevant conditions.

Platforms with nanoscale topography have recently become powerful tools in cellular biophysics and bioengineering. Recent studies have shown that nanotopography affects various cellular processes like adhesion and endocytosis, as well as physical properties such as cell shape. To engineer nanopillars more effectively for biomedical applications, it is crucial to gain better control and understanding of how nanopillars affect cell and nuclear physical properties, such as shape and spreading area, and impact cellular processes like endocytosis and adhesion. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate precise engineering of cellular architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, we discovered that cell elongation on nanopillars enhances nanopillar-induced endocytosis. These results have significant implications for various biomedical applications of nanopillars, including drug delivery, drug screening, intracellular electrophysiology, and biosensing. We believe our platform serves as a versatile tool for further explorations, facilitating investigations into the interplay between cell physical properties and alterations in cellular processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/543791v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b3a13dorg.highwire.dtl.DTLVardef@1edbdaorg.highwire.dtl.DTLVardef@1f3e40corg.highwire.dtl.DTLVardef@100d6fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Human bone marrow (BM) derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic and chondrogenic lineages enabling their application to clinical therapies. However, current methods, to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers, alone, can isolate a homogenous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications. TABLE OF CONTENTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/882563v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@48c5acorg.highwire.dtl.DTLVardef@1a9d096org.highwire.dtl.DTLVardef@1bd4da2org.highwire.dtl.DTLVardef@133dd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG