ACS Applied Bio Materials

Biomembrane coating technologies have increasingly been pursued to grant natural dynamic bio-interfaces onto synthetic nanomaterials. Herein, we report a one-step method to coat "living" biomembrane on nanoparticle surfaces in a non-destructive manner. In our method, nanoparticles were efficiently coated with cell membranes without losing the structural integrity by mechanically facilitating the passage of nanoparticles to a concentration layer of living cells with simple centrifugation. This was similar to the exosome-releasing process via endocytosis and exocytosis. The biomembrane originating from living Raw264.7 cells was coated onto the silica nanoparticle prepared by our method, and proteome profiling with nanoflow liquid chromatography-tandem mass spectrometry demonstrated that it was constructed with proteins derived from the membranous component. This proteome profile was not observed in silica nanoparticles prepared with dead cells. Finally, the hybridized cell membrane effectively suppressed the phagocytic activity of Raw264.7 cells to silica nanoparticles and improved the uptake efficiency into cancer cells. We believe our simple and efficient method to coat living biomembranes should be useful in developing medical and pharmaceutical applications involving nanoparticles.

The adhesive function of cell surface proteins can be visually assessed through direct observation; however, the underlying structures that mediate adhesion typically remain invisible at the nanoscale level. This hinders knowledge on the diversity of molecular architectures responsible for cell-cell adhesion. Drosophila E-cadherin (DE-cadherin), a classical cadherin with a unique domain structure, demonstrates adhesive function; however, it lacks a structural model that explains its adhesion mechanism. In this study, we present a novel application of DNA origami technology to create a cell-free, flat environment in which full DE-cadherin ectodomains are anchored using SNAP-tags and biotin-streptavidin interactions. DNA origami was assembled into a 120 nm long block, bearing 5 or 14 biotin:streptavidin sites that were evenly spaced on one lateral face. DE-cadherin ectodomain fragments were attached via biotinylated SNAP-tags. These decorated DNA origami nanoblocks were subjected to transmission electron and high-speed atomic force microscopy, which revealed a hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain, suggesting a role in mechanical flexibility. We also observed interactions between DE-cadherin ectodomains via their membrane-distal portions on single DNA origami nanoblocks. We reconstituted an adhesion-like process via pairing DNA origami nanoblocks using DE-cadherin ectodomain interactions. Homophilic associations of functional DE-cadherin ectodomains between the paired DNA origami nanoblocks were visualized at the nanoscale, displaying strand-like molecular configurations, likely representing the extracellular cadherin repeats without regular arrays of structural elements. This study introduces a DNA origami-based platform for reconstituting and visualizing cadherin ectodomain interactions, with potential applications for a broader range of adhesion molecules. HighlightsO_LIDNA origami technology was applied to perform a structure-function study of cadherin. C_LIO_LIDNA origami nanoblocks decorated with DE-cadherin ectodomains were observed by TEM/HS-AFM. C_LIO_LIA hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain was revealed. C_LIO_LIAn adhesion-like process was mimicked via pairing two nanoblocks using DE-cadherin ectodomain interactions. C_LIO_LIHomophilic associations of DE-cadherin ectodomains between the nanoblocks were visualized at the nanoscale level. C_LI

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.

Nanomaterials have been extensively investigated for their potential in delivering therapeutics to target tissues, but few have advanced to clinical application. The luminal surface of endothelial cells that line blood vessels are covered by a glycocalyx, a complex extracellular matrix rich in anionic glycans. However, the role of this glycocalyx in governing nanomaterial-cell interactions is often overlooked. In this study, we demonstrate that gold nanoparticles functionalized with branched polyethyleneimine (AuNP+) bind to primary human endothelial cells expressing either a developing or mature glycocalyx, with the interaction involving hyaluronan and heparan sulfate. Notably, the mature glycocalyx decreases the toxicity of AuNP+. In contrast, lipoic acid-functionalized gold nanoparticles (AuNP-) bind to endothelial cells with a developing glycocalyx, but not a mature glycocalyx. To further investigate this phenomenon, we studied charged polymers, including poly(arginine) (polyR) and poly(glutamic acid) (polyE). PolyE does not associate with endothelial cells regardless of glycocalyx maturity, but when glycans are enzymatically degraded, it can bind to the cells. Conversely, polyR associates with endothelial cells irrespective of glycocalyx maturity or glycan degradation. These findings highlight the intricate relationship between nanomaterial charge and presentation in interactions with endothelial cells, offering insights for modulating nanomaterial interactions with the blood vessel wall.

Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

The production of giant unilamellar vesicles (GUVs) plays a pivotal role in various scientific disciplines, particularly in the development of synthetic cells. While numerous methods exist for GUV preparation, the modified continuous droplet interface crossing encapsulation (cDICE) method offers the advantages of simplicity and high encapsulation efficiency. However, a significant limitation of this technique is the generation of vesicles with a broad size distribution and the inability to control the desired size range. This raises a key question: Can the modified cDICE method be optimized to produce GUVs with controlled size distribution? In this study, we examined the effects of two experimental parameters--rotation time (tROT) and the angular frequency ({omega}) of the cDICE chamber--on the size distribution of GUVs. Our results show that reducing either the angular frequency or rotation time shifts the size distribution toward larger vesicles, enabling effective size selection. These findings are further supported by a physical model, which provides insights into the mechanisms underlying size selection. This work demonstrates that control over GUV size distribution can be achieved through straightforward adjustments of system parameters. The ability to fine-tune vesicle size offers researchers a powerful tool for developing customizable experimental systems for synthetic biology and related fields.

Giant unilamellar vesicles (GUVs) embody biomimetic membranes with compartmentalization that serve not only as simplified models to better understand complex biochemical and biophysical processes, but also as a chassis for the bottom-up assembly of synthetic cells. Recently, double emulsion droplet microfluidics has proven to be a promising platform for their production, offering greater throughput, control, and reproducibility over traditional methods. However, the interplay of parameters--particularly under biocompatible conditions--that influence the complex multiphase fluid dynamics of the dewetting process underlying GUV production has not been thoroughly studied, limiting the democratization of the approach. In this study, we systematically investigate how lipid composition and concentration, aqueous phase conditions, droplet confinement, and fluid dynamics effects promote or impede the dewetting process. We show that the prevalent use of high concentrations of glycerol and P188 are unnecessary, and the altered dewetting dynamics with restricted surfactant usage can be tuned by adjusting chip dimensions and multi-phase compositions. Guided by these findings, we achieved robust, high throughput production of monodisperse GUVs using 0.1% P188 and no glycerol with salts. Our results improve the reliability and accessibility of droplet-microfluidics GUV platforms to catalyze advances in biophysics, synthetic biology, and drug discovery.

Nanoparticles (NPs) can be engineered to achieve targeted delivery with strategies based on surface modifications. These include layer-by-layer (LbL) NPs, modular electrostatically assembled carriers with tunable surface properties altered by changes to the outer polyion layer. Variations in these polymers dictate intracellular trafficking and biodistribution patterns. As NPs are administered, a layer of protein adsorbs to their surfaces, forming a protein corona that affects NP properties, alters biodistribution, and ultimately, impacts therapeutic efficacy. We hypothesized that some differences in LbL NP performance are due, in part, to variations in the resulting protein coronas. To study them, we first optimized an ultrafiltration method to effectively isolate LbL NPs with their protein corona. Following incubation in conditioned media, anionic homopolypeptide outer layers, such as poly-L-aspartic acid (PLD) and poly-L-glutamic acid (PLE), and LbL NPs with the bioinert polymer poly(acrylic acid) (PAA) had the lowest amount of protein associated, lower than conventional PEG liposomes. While mass spectroscopy revealed changes in the protein composition among LbL NPs; albumin, alpha-2-macroglobulin, and apolipoprotein B were most abundant. In vitro, pre-formed protein coronas reduced uptake in macrophages but increased uptake in ovarian cancer cells for certain LbL NP outer layers. In vivo, LbL NP outer layer influenced both serum half-life and biodistribution. Overall, this work highlights that LbL NPs can be designed to control protein corona formation, and supports that further understanding NP interactions with biological fluids is essential for designing clinically translatable NP platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/670086v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@18a2a66org.highwire.dtl.DTLVardef@1e5b755org.highwire.dtl.DTLVardef@676af2org.highwire.dtl.DTLVardef@19e3b34_HPS_FORMAT_FIGEXP M_FIG C_FIG

The widespread use of Covid-19 mRNA vaccines has highlighted the need to address rare but concerning side effects. Systemic off-target gene expression has been identified as a primary cause of acute adverse reactions and side effects associated with nucleoside-modified mRNA vaccines. In this study, we incorporated the permanent cationic lipid Dotap component into the mRNA-LNP formula associated with the FDA-approved mRNA vaccine Comirnaty to create a novel positively charged LNP carrier for mRNA vaccine delivery. Using the optimized LNP formula to prepare SARS-Cov-2 Spike mRNA vaccines for immunogenicity testing, Balb/c mice exhibited improved immunogenicity kinetics with initial antibody titers being lower but showing a continuous upward trend, ultimately reaching levels comparable to those of control mRNA vaccines 8 weeks after boost immunization. The mRNA vaccines encapsulated in the modified LNPs have demonstrated a superior safety profile in respect to systemic delivery of LNP constituents, off-target gene expression, and the systemic pro-inflammatory stimulation. Consequently, it may represent a safer alternative of conventional mRNA-LNP vaccines.

Pathogenic bacterial extracellular vesicles (BEVs) can disrupt the blood-brain barrier (BBB), leading to neuroinflammation. Prior in vitro studies of this process were performed in simple models that may have lacked important physiological factors. We sought to determine if treatment with Escherichia coli-derived BEVs could directly compromise the integrity of a BBB lab-on-chip model or if an immune component was required. Our device featured isogenic human induced pluripotent stem cell-derived brain microvascular endothelial-like cells (BMECs) and pericytes separated by an ultrathin, porous silicon nitride membrane. BEVs and free lipopolysaccharide (LPS) were capable of causing upregulation of intercellular adhesion molecule-1 on the BMEC surfaces, which is important for immune cell recruitment. However, neither BEVs nor LPS at physiological doses caused pronounced loss of BMEC tight junction proteins, nor did they increase barrier permeability to small dye molecules. In contrast, stimulating THP-1 macrophages with BEVs led to increased production of pro-inflammatory cytokines, and conditioned media from the stimulated macrophages disrupted BMEC tight junctions and increased barrier permeability. Our work demonstrates the importance of incorporating an immune component in studies of BEV-mediated disruption of BBB models.

In this paper, we report for the first time, the synthesis of a semiconducting biofilm. Photosynthetic bacterial biofilm has been used to weave together MoS2 nanosheets into an adherent film grown on interdigitated electrodes. Liquid-phase exfoliation of bulk MoS2 powder was used to obtain MoS2 nanosheets. A synchronous-fluorescence scan revealed the presence of two emission maxima at 682nm and 715nm for the MoS2 suspension. Such maxima with bandgap energy 1.82 and 1.73 eV corresponded to the single and double layer of MoS2. The presence of such single and multi-layered structures was confirmed by Raman spectroscopy, FTIR studies, and electron microscopy. The current-voltage (I-V) studies of such a bio-nano hybrid revealed the emergence of the gated nature of the current flow. This Schottky diode like behavior, reported earlier for Graphene-biofilm junctions, is also observed in this case. Gating voltage depended on the composition of the biofilm. The semiconductor biofilms, when studied using electrochemical impedance spectroscopy, revealed characteristic Nyquist and Bode plots, suggesting special circuit-equivalence for each film. While Mos2 was marked with stability with respect to variations in RMS voltage and bias voltage, the graphene biofilm was unique by the absence of any Warburg element.

Towards the goal of developing bio-chip / lab-on-a-chip substrates capable of performing highly specific bio-chemical reactions, Neutravidin binding to mixed Biotinylated Silane Self-Assembled Monolayers were studied using Confocal Fluorescence Light Microscopy. Non-specific bindings, specifically the formations of Neutravidin clusters, were quantified. Several experiments were conducted to determine the concentrations of Neutravidin necessary to not saturate surface binding to Biotinylated Self-Assembled Monolayers, determine the effectiveness of using FBS blocking buffers to reduce non-specific binding, optimize the repeatability of Neutravidin binding to Biotinlyated mixed Self-Assembled Monolayers with Silane-PEG-Biotin compositions ranging from 0 to 15%, and quantify background Neutravidin bindings and the corresponding formations of Neutravidin clusters to Self-Assembled Monolayers as Silane-PEG-Biotin percent compositions increase from 0 to 15%. The Neutravidin, Silane-PEG-Biotin, and Silane mPEG concentrations and ratios needed to develop homogeneous Neutravidin films, without the formations of clusters, on the Self-Assembled Monolayers have been determined.

Mucus is an impregnable barrier for drug delivery across the epithelia for treatment of mucosal-associated diseases. While current carriers are promising for mucus penetration, their surface chemistries do not possess chemical complexity to probe and identify optimal physicochemical properties desired for mucus penetration. As initial study, we use M13 phage display presenting random peptides to select peptides that can facilitate permeation through hyperconcentrated mucin. Here, a net-neutral charge, hydrophilic peptide was identified to facilitate transport of phage and fluorophore conjugates through mucin barrier compared to controls. This initial finding warrants further study to understand how composition and spatial distribution of physicochemical properties of peptides can be optimized to improve transport across the mucus barrier.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods, have drawbacks such as the requirement of high concentration in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/ml) of several target proteins into outer membrane vesicles (OMVs) of E. coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency arranging from 90-97% for different cell lines. Moreover, the free penetration of molecules less than 600 Dalton across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore physiologically how extracellular matrix components influence the cellular response. In this study, we aimed to use previously developed biomimetic streptavidin platforms to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, we observed that the interpretation of our findings was skewed due to the GAG-unrelated, non-specific adsorption of BMP2 on components of our biomaterials. Non-specific adsorption of proteins is a recurrent and challenging issue for biomaterial studies. Despite the initial incorporation of anti-fouling poly(ethylene glycol) (PEG) chains within our biomaterials, the residual non-specific BMP2 adsorption still triggered BMP2 signaling within the same range as our conditions of interest. To tackle this issue, we explored various options to prevent BMP2 non-specific adsorption. Specifically, we tested alternative constructions of our biomaterials on gold or glass substrate using distinct PEG-based linkers. We identified the aggregation of BMP2 at neutral pH as a potential cause of non-specific adsorption and thus determined specific buffer conditions to prevent it. We also investigated the induced BMP2 signaling over different culture periods. Nevertheless, none of these options resulted in a viable suitable solution to reduce the non-specific BMP2 signaling. Next, we studied the effect of various blocking strategies. We identified a blocking condition involving a combination of bovine serum albumin and trehalose that successfully reduced the unspecific attachment of BMP2 and the non-specific signaling. Furthermore, the effect of this blocking step was improved when using gold platforms instead of glass, particularly with Chinese hamster ovary (CHO) cells that seemed less responsive to non-specifically bound BMP2 than C2C12 cells.

Proteins can alter their shape when interacting with a surface. This study explores how bovine serum albumin (BSA) modifies structurally when it adheres to a gold surface, depending on the protein concentration and pH. We verified that the gold surface induces significant structural modifications to the BSA molecule using circular dichroism, infrared spectroscopy, and atomic force microscopy. Specifically, adsorbed molecules displayed increased levels of disordered structures and {beta}-turns, with fewer -helices than the native structure. MP-SPR spectroscopy demonstrated that the protein molecules preferred a planar orientation during adsorption. Molecular dynamics simulations revealed that the interaction between cysteines exposed to the outside of the molecule and the gold surface was vital, especially at pH = 3.5. The macroscopic properties of the protein film observed by AFM and contact angles confirm the flexible nature of the protein itself. Notably, structural transformation is joined with the degree of hydration of protein layers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/567678v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@525beborg.highwire.dtl.DTLVardef@110d1adorg.highwire.dtl.DTLVardef@135eed9org.highwire.dtl.DTLVardef@1d3ec41_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fungal adhesion to stainless steel, an alloy commonly used in food and beverage sectors, public and healthcare settings, and numerous medical devices, can give rise to serious infections, ultimately leading to morbidity, mortality, and significant healthcare expenses. In this study, we demonstrate that nanotextured stainless steel (nSS) fabricated using an electrochemical technique is an antibiotic-free biocidal surface against Candida Albicans and Fusarium Oxysporum with 98% and 97% reduction, respectively. The nanoprotrusion features on nSS can have both physical contact with cell membranes and chemical impact on cells through production of reactive species, this material should not contribute to drug resistant fungus as antibiotics can. As nSS is also antibacterial and compatible with mammalian cells, demonstration of antifungal activity gives nSS the potential to be used to create effective, scalable, and sustainable solutions to broadly and responsibly prevent fungal and other microbial infections caused by surface contamination. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/616307v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9c037dorg.highwire.dtl.DTLVardef@a93518org.highwire.dtl.DTLVardef@dccc6forg.highwire.dtl.DTLVardef@1f19515_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

AbstractsThe ideal engineered microbial smart-drug should be capable of functioning on demand at specific sites in vivo. However, precise regulation of engineered microorganisms poses challenges in the convoluted and elongated intestines. Despite the promising application potential of optogenetic regulation strategies based on light signals, the poor tissue penetration of light signals limits their application in large experimental animals. Given the rapid development of ingestible electronic capsules in recent years, taking advantage of them as regulatory devices to deliver light signals in situ to engineered bacteria within the intestines has become feasible. In this study, we established an electronic-microorganism signaling system, realized by two Bluetooth-controlled luminescent electronic capsules were designed. The "Manager" capsule is equipped with a photosensor to monitor the distribution of engineered bacteria and to activate the optogenetic function of the bacteria by emitting green light. The other capsule, "Locator", can control the in situ photopolymerization of hydrogels in the intestines via ultraviolet light, aiding in the retention of engineered bacteria at specific sites. These two electronic capsules are expected to work synergistically to regulate the distribution and function of engineered bacteria in vivo, and their application in the treatment of colitis in pigs is currently being investigated, with relevant results to be updated subsequently.

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.