Soft Electroporation Through 3D Hollow Nanoelectrodes.

Generally, electroporation of in vitro cells is performed under very high electric fields to overcome the physical barrier of plasma membrane. Since traditional electroporation techniques make use of very high voltages, which is critical to cell viability, this study presents a microfluidic platform able to perform cell membrane electroporation with the application of low voltages (1.5-2 V). The platform is manufactured based on the milling by mean of focused ionic beam, which offers an established approach to fabricate ordered arrays of 3D gold hollow nanoelectrodes protruding from an insulating substrate. The novelty of this fabrication relies on the fact that the nanoelectrodes used for electroporation are simultaneously metallic, hollow and communicate through its nanochannels with an isolated microfluidic chamber beneath the device. Adherent cultured cells on the nanoelectrodes can be electroporated in this platform, and molecules can be selectively delivered only inside the porated cells.

The Bio-Safety Concerns of Three Domestic Temporary Hair Dye Molecules: Fuchsin Basic, Victoria Blue B and Basic Red 2.

Hair-coloring products include permanent, semi-permanent and temporary dyes that vary by chemical formulation and are distinguished mainly by how long they last. Domestic temporary hair dyes, such as fuchsin basic, basic red 2 and Victoria blue B, are especially popular because of their cheapness and facile applications. Despite numerous studies on the relationship between permanent hair dyes and disease, there are few studies addressing whether these domestic temporary hair dyes are associated with an increased cancer risk. Herein, to ascertain the bio-safety of these temporary hair dyes, we comparatively studied their percutaneous absorption, hemolytic effect and cytotoxic effects in this paper. Furthermore, to better understand the risk of these dyes after penetrating the skin, experimental and theoretical studies were carried out examining the interactions between the dyes and serum albumins as well as calf thymus (CT)-DNA. The results showed that these domestic temporary hair dyes are cytotoxic with regard to human red blood cells and NIH/3T3 cell lines, due to intense interactions with bovine serum albumin (BSA)/DNA. We conclude that the temporary hair dyes may have risk to human health, and those who use them should be aware of their potential toxic effects.

5-Azacytidine specifically inhibits the NIH-3T3 PCD process induced by TNF-alpha and cycloheximide via affecting BCL-XL.

DNA methylation plays a crucial role in lots of biological processes and cancer. 5-azacytidine (5-AC), a DNA methylation inhibitor, has been used as a potential chemotherapeutic agent for cancer. In this study, we used 5-AC treatment to investigate whether DNA methylation was involved in regulation of programmed cell death (PCD) in mouse embryo fibroblast NIH-3T3 cells which could undergo PCD after treatment with TNF-α and cycloheximide (CHX). The results showed that the genomic DNA of NIH-3T3 cells was hypermethylated during PCD induced by TNF-α and CHX, and 5-AC might prevent this PCD process. However, treatment with the other three DNA methylation inhibitors, 5-aza-deoxycytidine, 6-thioguanine and RG108, did not interfere with the NIH-3T3 cell PCD process. Additionally, knockdown of DNMT1 did not affect the apoptosis process. The present results and observations indicated that 5-AC specifically inhibited the NIH-3T3 apoptosis process via a genomic DNA methylation-independent pathway. During the TNF-α and CHX-inducing apoptosis process, the PCD related BCL-2 family proteins were significantly down-regulated. Furthermore, after the small interference RNA-mediated knockdown of BCL-XL, one of the BCL-2 family proteins, 5-AC did not inhibit the apoptosis process, suggesting that 5-AC inhibited the PCD process induced by TNF-α and CHX by affecting the anti-apoptotic protein BCL-XL.

Regulation of nuclear morphology by actomyosin components and cell geometry.

Extracellular microenvironmental signals modulate the coupling between cytoskeleton to nuclear links to regulate gene expression profiles. However the influence of actomyosin assembly on the morphology of the nucleus is not well understood. In this paper, we quantitatively demonstrate the role of cell geometry and specific actomyosin molecular components in their control of nuclear morphology.

IRSp53 mediates podosome formation via VASP in NIH-Src cells.

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.

Controlled protein absorption and cell adhesion on polymer-brush-grafted poly(3,4-ethylenedioxythiophene) films.

Tailoring the surface of biometallic implants with protein-resistant polymer brushes represents an efficient approach to improve the biocompability and mechanical compliance with soft human tissues. A general approach utilizing electropolymerization to form initiating group (-Br) containing poly(3,4-ethylenedioxythiophen)s (poly(EDOT)s) is described. After the conducting polymer is deposited, neutral poly((oligo(ethylene glycol) methacrylate), poly(OEGMA), and zwitterionic poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(SBMA), brushes are grafted by surface-initiated atom transfer radical polymerization. Quartz crystal microbalance (QCM) experiments confirm protein resistance of poly(OEGMA) and poly(SBMA)-grafted poly(EDOT)s. The protein binding properties of the surface are modulated by the density of polymer brushes, which is controlled by the feed content of initiator-containing monomer (EDOT-Br) in the monomer mixture solution for electropolymerization. Furthermore, these polymer-grafted poly(EDOT)s also prevent cells to adhere on the surface.

Generation of mechanical and biofunctional gradients in PEG diacrylate hydrogels by perfusion-based frontal photopolymerization.

The spatial presentation of soluble growth factors, immobilized extracellular matrix molecules, as well as matrix rigidity, plays an important role in directed and guided cell migration. Synthetic hydrogel scaffolds offer the ability to systematically introduce gradients of these factors contributing to our understanding of how the 3D arrangement of biochemical and mechanical cues influence cell behavior. Using a novel photopolymerization technique, perfusion-based frontal photopolymerization (PBFP), we have engineered poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with gradients of mechanical properties and immobilized biofunctionality. The controlled delivery of a buoyant photoinitiator, eosin Y, through a glass frit filter results in the formation and subsequent propagation of a polymer reaction front that is self-sustained and able to propagate through the monomeric mixture. Propagation of this front results in monomer depletion, leading to variations in cross-linking, as well as spatial gradients of elastic modulus and immobilized concentrations of the YRGDS cell adhesion ligand within PEGDA hydrogels. Furthermore, the magnitudes of the resulting gradients are controlled through alterations in polymerization conditions. Preliminary in vitro cell-culture studies demonstrate that the gradients generated stimulate directed 2D cell growth on the surface of PEGDA hydrogels. By day 14, fibroblast aggregates spread roughly twice as far in the direction parallel to the slope of the gradient as compared to the perpendicular direction. The presented technique has great potential in controlling gradients of mechanical properties and immobilized biofunctionality for directing and guiding 3D cell behavior within tissue-engineered scaffolds.

Fibrous scaffolds made by co-electrospinning soluble eggshell membrane protein with biodegradable synthetic polymers.

Soluble eggshell membrane protein (SEP), isolated from natural eggshell membrane, was co-electrospun with biodegradable synthetic polymers poly(propylene carbonate) (PPC) and poly(lactic acid) (PLA) in various proportions from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions in order to prepare fibrous scaffolds having simultaneously good mechanical properties and biocompatibility. The fiber morphology was observed by field emission scanning electron microscopy, showing uniform fibers with diameter of 1.2-1.0 and 1.3-0.7 um for PPC/SEP and PLA/SEP blend fibers, respectively. Transmission electron microscopy observation shows that the blend fibers have domain-matrix phase morphology with fiber-like SEP domains in the PPC or PLA matrix, indicating the occurrence of phase separation, although interaction exists between PPC (or PLA) and SEP, as revealed by attenuated total reflectance Fourier transform infrared spectroscopy. The mechanical properties were evaluated by uniaxial tensile tests and showed that both the tensile strength and elongation at break increase with increasing incorporation of PPC (or PLA). The surface composition was investigated by X-ray photoelectron spectroscopy and SEP was found on the fiber surfaces, and as a result the surfaces of the fibrous scaffolds are superhydrophilic. NIH3T3 cell culture tests demonstrate that the PPC/SEP and PLA/SEP blend fibrous scaffolds have a much improved biocompatibility compared to pure PPC or PLA fibrous scaffolds.

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling.

BACKGROUND: Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells. RESULTS: Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2) domain of Hck (Hck-SH2) was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet)-Hck-SH2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet) and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. CONCLUSION: These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

Cyto- and hemocompatibility of a biodegradable 3D-scaffold material designed for medical applications.

In this study, the polyester urethane Degrapol (DP) was explored for medical applications. Electrospun DP-fiber fleeces were characterized with regard to fiber morphology, swelling, and interconnectivity of interfiber spaces. Moreover, DP was assayed for cell proliferation and hemocompatibility being a prerequisite to any further in vivo application. It was shown that DP-fiber fleeces produced at different humidity while spinning affects interconnectivity of interfiber spaces, such that the higher the humidity the looser the resulting fiber fleeces. When the spinning target was cooled with dry ice, the resulting DP-fibers remained less fused to each other. However, permeability for fluorescent beads was not significantly increased. Fibroblast adhesion and proliferation occurred in a comparable manner on native as well as on fibronectin or collagen I adsorbed DP-fiber fleeces. On DP-surfaces fibroblasts proliferated equally well as compared with glass or PLGA surfaces or DP-surfaces adsorbed with fibronectin or collagen I. In contrast, human umbilical vein endothelial cells proliferated only after adsorption of DP-surfaces with fibronectin or collagen I, indicating that different cell types respond differently to DP-surfaces. Furthermore, hemocompatibility of DP-surfaces was found to be similar or better to PLGA or stainless steel, both medically used materials. These experiments indicate that DP-fiber fleeces or surfaces might be useful for tissue engineering.

The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells.

The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

Controlling the mechanics and nanotopography of biocompatible scaffolds through dielectrophoresis with carbon nanotubes.

Creating a biocompatible carbon-nanotube polymer scaffold is an area that has a number of potential applications. Herein, a dielectrophoretic approach was pursued to integrate carbon nanotubes into a polymeric material for fabricating a nanoscale composite scaffold with increased and controllable mechanical strength. The adhesion force, which combines the surface energy of the sample and the interfacial energy between the tip and sample, was estimated to be 55.39 +/- 6.72 nN away from the center of the protrusions at a distance of 0.5 microm while being 24.01 +/- 4.45 nN at the center. The adhesion force for the center of the cavities was 42.47 +/- 6.91 and 88.21 +/- 15.05 nN at 0.5 microm away from the center. NIH 3T3 fibroblast cells were then utilized to test the cellular biocompatibility of this multiwalled carbon nanotubes (MWCNTs) film. Cells were cultured on the surface and then their attachment, spreading, and proliferation behaviors were observed. This nanotube-polymer scaffolding approach has a wide range of potential applications including in complex device fabrication as well as in developing biomimetic and tissue engineering scaffolds, and artificial organs.

A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations.

Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.

HLA-B35 influences the apoptosis rate in human peripheral blood mononucleated cells and HLA-transfected cells.

Human leukocyte antigen (HLA) class I antigens can act as signal-transducing molecules that influence individual reactivity to external stimuli and the existence of haplotype-specific cell signal regulation has been suggested. In this article, we provide definite experimental evidence for the existence of a HLA-B35 haplotype-specific regulation of cell apoptosis in different experimental models. First, we demonstrated that HLA-B35, but not other HLA-class I antigens, was associated with an increased cell susceptibility to apoptosis in human peripheral mononuclear cells (PBMCs) exposed in vitro to thapsigargin. Second, we confirmed this association in human ECV 304 cells transfected with HLA-B35 or with HLA-B8, an antigen that did not appear to influence the apoptosis rate in the thapsigargin-treated PBMCs. Third, we confirmed the specific influence of HLA-B35 on cell apoptosis in non human cells (i.e., HLA-B35-transfected NIH3T3 murine fibroblasts). Our data show the existence of HLA-B35 haplotype-specific regulation of cell apoptosis and open new perspectives on the role of HLA class I genes in cell activation and disease susceptibility.

Epigenetic activation of a subset of mRNAs by eIF4E explains its effects on cell proliferation.

BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5' cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction.

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.

Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts.

Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca(2+)-independent phospholipase A(2) (iPLA(2)) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA(2)-IVA, cPLA(2)-IVB, cPLA(2)-IVC, iPLA(2)-VIA, iPLA(2)-VIB, and secretory sPLA(2)-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA(2)-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA(2)-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA(2) substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA(2) activity was also detected in lysates devoid of sPLA(2), indicating that both sPLA(2) and iPLA(2) contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA(2) and possibly also by sPLA(2), whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA(2) and, to a lesser extent, iPLA(2).

Cytotoxic and mutagenic effects of tobacco-borne free fatty acids.

Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a]pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking.

Characterization of PMP22 expression in osteosarcoma.

The peripheral myelin protein (PMP22) gene is highly expressed in peripheral Schwann cells and encodes an important constituent of the myelin sheath. It is also expressed at lower levels in other normal tissues in which the protein is supposed to be involved in cell growth regulation. We recently reported frequent amplification and overexpression of PMP22 in high-grade osteosarcoma. Here, we analyzed PMP22 expression in five osteosarcoma tumors and three osteosarcoma cell lines. In normal Schwann cells, transcription of PMP22 starts at three promoters, P1A, P1B, and P2, which results in the synthesis of three alternatively spliced transcripts that all code for the same protein. We found a comparable expression pattern in normal osteoblasts. However, promoter P1A-driven transcripts were absent in all investigated tumors and cell lines and, compared to normal osteoblasts, the P1B/P2 transcript ratio was found to be increased in two of three cases with PMP22 overexpression and decreased in all five cases without overexpression. In normal Schwann cells and in NIH3T3 cells, PMP22 expression increases upon serum starvation-induced growth arrest. In contrast to this, serum withdrawal caused a considerable decrease of PMP22 expression in the osteosarcoma cell lines. We conclude that the different PMP22 expression in osteosarcoma may result in alternative availability of the PMP22 protein during the cell cycle and aberrant regulation of cell growth control in osteosarcoma tumorigenesis.

Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer.

Structural changes of proteins during adsorption to biomaterials affect the presentation of molecular binding sites and, ultimately, biomaterial performance. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study structural changes of the cell adhesion protein, fibronectin (Fn), following adsorption to model hydrophilic and hydrophobic surfaces. Fn was labeled with donor and acceptor fluorophores using two labeling schemes and intramolecular energy transfer was calibrated against measured structural changes of Fn in denaturing solutions. FRET was then applied to measure Fn's structure on surfaces. Based on FRET, Fn underwent greater extension of its dimer arms on hydrophilic glass than on hydrophobic fluoroalklysilane-derivatized glass (fluorosilane), and this extension was insensitive to molecular packing over a range of adsorption concentrations. Fn's conformation on glass better promoted cell attachment than on fluorosilane; the roles of both global structural changes (movements of modules) and local structural changes (disruption of secondary structure) on Fn's cell integrin binding activity are discussed. Based on previous FRET work, we compare Fn's conformations on these surfaces with its conformations in fibroblast culture. FRET is unique in allowing direct comparison of protein structure between biomaterial surfaces and cell culture.