Model Biomaterial Surfaces Research Articles

RETRACTED: Assessment of antibiofilm activity of magnesium fluoride nanoparticles-stabilized oil-in-water nanosized emulsion

RETRACTED

Fibroblasts remodeling of type IV collagen at a biomaterials interface.

This paper describes the fate of adsorbed type IV collagen (Col IV) in contact with fibroblasts on model biomaterial surfaces, varying in wettability, chemistry and charge. We found that fibroblasts not only interact but also tend to remodel differently adsorbed Col IV employing two distinct mechanisms: mechanical reorganization and proteolytic degradation. Apart from the trend of adsorption -NH2 > CH3 > COOH > OH- the cells interact better with NH2 and OH surfaces -i.e. independently of the amount of adsorbed Col IV - evident from the quantitative measurements of cell adhesion and spreading and the improved recruitment of alpha 1 and alpha 2 integrins as well as p-FAK in focal adhesions. The linearly arranged Col IV co-localize with FN fibrils formed from either secreted, or exogenously added protein, which confirms their interdependence during a reorganization process. We further found that this reorganization is better pronounced on hydrophilic OH and positively charged NH2 surfaces correlating with the improved cellular interaction. Conversely, the fibroblasts tend to round on COOH and CH3 surfaces in compliance with the altered integrin signaling and also the increased pericellular proteolysis activity quantified by the increased de-quenching of adsorbed FITC-Col IV and zymography. Taken together, these results show that remodeling of Col IV at a cell-biomaterial interface depends strongly on the surface properties of a material and affects significantly its biological performance.

Characterization of Biomaterials by Soft X-Ray Spectromicroscopy.

Synchrotron-based soft X-ray spectromicroscopy techniques are emerging as useful tools to characterize potentially biocompatible materials and to probe protein interactions with model biomaterial surfaces. Simultaneous quantitative chemical analysis of the near surface region of the candidate biomaterial, and adsorbed proteins, peptides or other biological species can be obtained at high spatial resolution via scanning transmission X-ray microscopy (STXM) and X-ray photoemission electron microscopy (X-PEEM). Both techniques use near-edge X-ray absorption fine structure (NEXAFS) spectral contrast for chemical identification and quantitation. The capabilities of STXM and X-PEEM for the analysis of biomaterials are reviewed and illustrated by three recent studies: (1) characterization of hydrophobic surfaces, including adsorption of fibrinogen (Fg) or human serum albumin (HSA) to hydrophobic polymeric thin films, (2) studies of HSA adsorption to biodegradable or potentially biocompatible polymers, and (3) studies of biomaterials under fully hydrated conditions. Other recent applications of STXM and X-PEEM to biomaterials are also reviewed.

Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers

Polyelectrolyte multilayer coatings have emerged as substrates to control a variety of cell behaviour, including adhesion, proliferation and differentiation. In particular, it is possible to modulate film stiffness by physical or chemical cross-linking. In this study, we evaluate the adhesive behaviour of skeletal muscle cells (C2C12 myoblasts) during the initial steps of spreading on layer-by-layer films of controlled stiffness made of poly(L-lysine) and hyaluronan as model biomaterial surfaces for muscle tissue engineering. We show that integrin clustering, integrin actin cytoskeleton connection and focal adhesion formation for cell spreading can be decoupled by controlling film stiffness. This made it possible to switch the cells morphologically between round and spreading shapes depending on the stiffness of the microenvironment. Although hyaluronan is one of the main components of cross-linked multilayer films, the HA receptor CD44 did not appear to mediate early adhesion as suggested by the use of blocking antibodies. In contrast, integrins were found to play a pivotal role in early adhesion: their activation significantly enhanced C2C12 myoblast spreading on soft films, where they were otherwise round. Integrin clustering was also induced by the softer films and enhanced on the stiffest films. Conversely, the use of soluble inhibitors or blocking antibodies directed against integrins induced a round phenotype on stiff films, where cells were well spread out in control conditions. We show that specific integrins were involved in the adhesion process as blocking β(3), but not β(1), integrins inhibited cell adhesion. These soft, stiff films can thus be used to tune the adhesion of C2C12 myoblasts, an early key event in myogenesis, via integrin clustering and subsequent signalling. They may be further used to decorticate the signalling pathways associated with β(3) integrins.

Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides

The aim of this study was to evaluate a new approach to inhibit complement activation triggered by biomaterial surfaces in contact with blood. In order to inhibit complement activation initiated by the classical pathway (CP), we used streptococcal M protein-derived peptides that specifically bind human C4BP, an inhibitor of the CP. The peptides were used to coat polystyrene microtiter wells which served as a model biomaterial. The ability of coated peptides to bind C4BP and to attenuate complement activation via the CP (monitored as generation of fluid-phase C3a and binding of fragments of C3 and C4 to the surface) was investigated using diluted normal human serum, where complement activation by the AP is minimal, as well as serum from a patient lacking alternative pathway activation. Complement activation (all parameters) was significantly decreased in serum incubated in well surfaces coated with peptides. Total inhibition of complement activation was obtained at peptide coating concentrations as low as 1–5 μg/mL. Successful use of Streptococcus-derived peptides shows that it is feasible to control complement activation at a model biomaterial surface by capturing autologous complement regulatory molecules from plasma.

Real-Time Monitoring of Fibrinogen Cross-Linking on Model Biomaterial Surfaces with Quartz Crystal Microbalance

A quartz crystal microbalance was used for real-time monitoring of fibrinogen cross-linking on three model biomaterial surfaces. Fibrinogen adsorbs slowly and forms a less rigid multi-layer on hydrophobic surfaces, while it adsorbs quickly, forming a single mono-layer on hydrophilic surfaces. The extent of fibrinogen cross-linking is greater on hydrophobic surfaces. Fibrinogen cross-linking can also rigidify the relatively soft coatings of poly(methyl methacrylate) and dodecanethiol self-assembled monolayer.

In situ photoelectron spectroscopy study of water adsorption on model biomaterial surfaces

Using in situ photoelectron spectroscopy at near ambient conditions, we compare theinteraction of water with four different model biomaterial surfaces: self-assembled thiolmonolayers on Au(111) that are functionalized with methyl, hydroxyl, and carboxyl groups,and phosphatidylcholine (POPC) lipid films on silicon. We show that the interaction ofwater with biomaterial surfaces is mediated by polar functional groups that interactstrongly with water molecules through hydrogen bonding, resulting in adsorption of0.2–0.3 ML water on the polar thiol films in 700 mTorr water pressure and resulting incharacteristic N 1s and P 2p shifts for the POPC films. Provided that beam damage iscarefully controlled, in situ electron spectroscopy can give valuable informationabout water adsorption which is not accessible under ultrahigh vacuum conditions.

Surface modification of biomaterials to control adhesion of cells

The layer-by-layer technique was used to build-up polyelectrolyte multilayers (PEMs) composed of heparin, an anionic glycosaminoglycan (GAG) and chitosan, a cationic biodegradable polysaccharide on model biomaterial surfaces. The surface coatings shall control adhesion of cells and thus their subsequent proliferation and differentiation. PEMs were characterized physicochemically by static contact angle and quartz crystal microbalance (QCM) measurements. Variations in procedure parameters such as the pH value of the solutions were crucial to the formation process and surface properties in terms of wettability and mass increase. Cell-surface interactions were studied with human fibroblast on PEMs. It was found that the pH value of solutions had a strong impact on cell adhesion making surfaces extremely cytophobic or moderately cytophilic. Adsorption of fibronectin to the terminal heparin layer could be used to increase cell adhesion in a concentration-dependent manner.

Rho GTPase protein expression and activation in murine monocytes/macrophages are not modulated by model biomaterial surfaces in serum-containing in vitro cultures

The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue-culture polystyrene, polystyrene, poly-L-lactide and Teflon® AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue-culture polystyrene and poly-L-lactide surfaces after several days, but absent from those grown on apolar polystyrene and Teflon® AF, indicating some surface influence on this GTPase in serum-containing cultures.

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH(2)-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH(3) groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against beta(1) integrin inhibited matrix mineralization on the OH and NH(2) surfaces, whereas function-perturbing antibodies specific for beta(3) integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.

Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface

In the present study we investigate whether complement activation in blood in contact with a model biomaterial surface (polystyrene) occurs directly on the material surface or on top of an adsorbed plasma protein layer. Quartz crystal microbalance-dissipation analysis (QCM-D) complemented with enzyme immunoassays and Western blotting were used. QCM-D showed that the surface was immediately covered with a plasma protein film of approximately 8 nm. Complement activation that started concomitantly with the adsorption of the protein film was triggered by a self-limiting classical pathway activation. After adsorption of the protein film, alternative pathway activation provided the bulk of the C3b deposition that added 25% more mass to the surface. The build up of alternative pathway convertase complexes using purified C3 and factors B and D on different protein films as monitored by QCM-D showed that only adsorbed albumin, IgG, but not fibrinogen, allowed C3b binding, convertase assembly and amplification. Western blotting of eluted proteins from the material surface demonstrated that the C3 fragments were covalently bound to other proteins. This is consistent with a model in which the activation is triggered by initiating convertases formed by means of the initially adsorbed proteins and the main C3b binding is mediated by the alternative pathway on top of the adsorbed protein film.

Molecular-Scale Studies of Proteins at Model Biomaterial Surfaces by Atomic Force Microscopy

A widely accepted tenet of biomaterials research is that the initial step following contact of a synthetic material with blood is the rapid adsorption of plasma proteins. The composition of this adsorbed protein layer is dependent on a variety of factors, including the surface properties of the implant material and the nature of the adsorbing proteins, and the composition and function of this protein layer is important in the subsequent biological response and ultimately the success or failure of the implanted material. While a great amount of effort has gone into developing structure/function responses for implanted biomaterials, there is still much about the molecular level interactions to be determined. We utilized atomic force microscopy (AFM) to investigate the molecular-level interactions of proteins with model biomaterial substrates. The AFM is unique in that it offers the opportunity to characterize interfacial environments, determine material properties, measure protein/surface interaction forces, and visualize the tertiary structure of adsorbed proteins.

Binding of a model regulator of complement activation (RCA) to a biomaterial surface: surface-bound factor H inhibits complement activation

The complement system is an important inflammatory mediator during procedures such as cardiopulmonary bypass and hemodialysis when blood is exposed to large areas of biomaterial surface. This contact between blood and the biomaterials of implants and extracorporeal circuits leads to an inflammatory response mediated by the complement system. The aim of this study was to assess the ability of a complement regulator (factor H) immobilised on a biomaterial surface to inhibit complement cascade mediated inflammatory responses. The cross-linker N-succinimidyl 3-(2-pyridyldithio) propionate was used to immobilise factor H on a model biomaterial surface without affecting the biological activity of the inhibitor. Binding of factor H was then characterised using quartz crystal microbalance-dissipation (QCM-D) and enzyme immunoassays for products of complement activation: bound C3 fragments and soluble C3a, sC5b-9, and C1s-C1INA. Immobilised factor H reduced the amount C3 fragments deposited on the biomaterial surface after incubation with serum, plasma, or whole blood. In addition, lower levels of soluble C3a and sC5b-9 were generated after incubation with whole blood. In summary, we have demonstrated that complement activation on a highly activating model surface can be inhibited by immobilised factor H and have defined prerequisites for the preparation of future biomaterial surfaces with immobilised regulators of complement activation.

BIOLOGICAL INTERACTIONS WITH POLYMER BIOMATERIALS EVALUATED AT MICROSCOPIC AND NANOSCOPIC LEVELS

Polymer biomaterials are used in a variety of blood-contacting medical devices. In particular, poly(urethaneurea) (PUU) based biomaterials have proven well-suited for devices due to their good mechanical properties and superior blood compatibility compared to other synthetic materials. This has largely been attributed to the phenomena of microphase separation, as the PUU copolymer separates into distinct “hard” and “soft” microphases. Although a variety of evidence relating microphase separation and blood compatibility exists in the literature, there is no direct evidence relating the two phenomena at a molecular level. We have attempted to leam more about the biological interactions with polymer biomaterials including PUU materials by utilizing high resolution fluorescence microscopy and atomic force microscopy (AFM). AFM was used to characterize the microphase structure of a series of PUU films of varying hard segment/soft segment ratio and to determine the interactions of proteins with synthetic polymer materials under aqueous buffer conditions. Fluorescence microscopy with immunohistochemical staining was used to study platelet interactions with PUU materials under in-vitro conditions as well as protein interactions with PUU blood sacs retrieved from total artificial heart implanted animals. AFM images demonstrated a characteristic microphase structure in the PUU films best described as randomly oriented cylinders with occasional spherical domains. High-resolution studies of individual plasma proteins on hydrophobic polymer materials reveal tertiary structures consistent with what others have observed previously on ultrasmooth model biomaterial surfaces. The fluorescence microscopy images demonstrated protein deposition aligned with the expected direction of blood flow in the implanted devices. Taken together, these data provide insight into the nature of biomaterial/biological interactions under physiologically relevant conditions.

Biomimetic peptide surfaces that regulate adhesion, spreading, cytoskeletal organization, and mineralization of the matrix deposited by osteoblast-like cells.

In an effort to regulate mammalian cell behavior in contact with solid material surfaces, we have functionalized surfaces with different ratios of both the putative cell binding (-Arg-Gly-Asp-) domain and a consensus heparan-binding domain. The peptide sequences -Arg-Gly-Asp- (-RGD-) and -Phe-His-Arg-Arg-Ile-Lys-Ala- (-FHRRIKA-) or mixtures of the two in the ratios of 75:25 (mimetic peptide surface I), 25:75 (mimetic peptide surface II), and 50:50 (mimetic peptide surface III) were immobilized on model surfaces using a heterobifunctional cross-linker to link the peptide(s) to amine-functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry, thickness of the overlayers, and surface density of immobilized peptides ( approximately 4-6 pmol/cm2). The degree of rat calvaria osteoblast-like cell spreading, focal contact formation, cytoskeletal organization, proliferation, and mineralization of the extracellular matrix (ECM) on model biomaterial surfaces was examined. Mimetic peptide surface II (MPS II) and MPS III supported the highest degree of cell spreading (p < 0.05), following 4 h of incubation, compared to MPS I, homogeneous -RGD-, and homogeneous -FHRRIKA- grafted surfaces. Furthermore, MPS I, MPS II, MPS III, and homogeneous -RGD- surfaces promoted the formation of focal contacts and stress fibers by attached bone cells. The strength of bone cell detachment following 30 min of incubation was significantly higher (p < 0.05) on MPS II surfaces compared to homogeneous -RGD- and -FHRRIKA-. However, the degree of cell proliferation on the peptide surfaces were not significantly different from each other (p > 0.1). Following 24 d in culture, the areas of mineralized ECM formed on MPS II and MPS III surfaces were significantly (p < 0.05) larger than those of other surfaces. These results demonstrate that utilizing peptide sequences incorporating both cell- and heparin-adhesive motifs can enhance the degree of cell surface interactions and influence the long-term formation of mineralized ECM in vitro.

Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides

The process of cell migration is inextricably linked with the process of cell adhesion and, therefore, with cell/substrate adhesiveness. The present study adapted an under-agarose cell migration assay to quantitatively examine population migration characteristics of osteoblasts, on substrates modified with adhesive peptides, in the absence and presence of growth factors. Short-term, that is, 48 h osteoblast migration distances on substrates modified with adhesive Arg–Gly–Asp–Ser peptides were significantly ( P<0.05) less than migration distances on substrates modified with non-adhesive Arg–Asp–Gly–Ser peptides, demonstrating that osteoblast population haptokinesis was significantly decreased on substrates modified with adhesive peptides. Random motility coefficients calculated in the present study for osteoblast populations were an order of magnitude lower than a published random motility coefficient for leukocytes, proving quantitatively that, compared to leukocytes, osteoblasts migrate via haptokinesis more slowly. The 48 and 72 h osteoblast population migration differentials in the presence of an initial mass of 60 ng of basic Fibroblast Growth Factor, on substrates modified with Arg–Gly–Asp–Ser or with Arg–Asp–Gly–Ser, were larger than all other chemotactic differentials on these substrates. Quantitative investigations (such as the present study) of cell population migration characteristics on model biomaterial surfaces will become increasingly necessary as the discipline of cell/tissue engineering matures.

Nanostructured model biomaterial surfaces prepared by colloidal lithography

Adsorption of 110 nm negatively charged polystyrene particles on positively charged titanium surfaces was studied for the purpose of making surfaces with controlled nanotopography. Surfaces with 9–26 % saturation coverages of evenly spaced particles were produced by varying the salt concentration in the particle solution (0.01–1 mM NaCl). This is a quick and relatively simple method to make uniform nanostructures over large surface areas (~cm 2). The produced surfaces are used for in vitro cell studies of how sub-micrometre topography influences cell adhesion and function.

Complement activation and inflammation triggered by model biomaterial surfaces.

Biomaterial-mediated complement activation repeatedly has been invoked as a trigger of phagocyte reactions and inflammation. However, a direct correlation between complement activation and inflammatory responses to biomaterial surfaces has yet to be established. Using an animal implantation model and gold surfaces bearing various thiol-linked functionalities, we investigated the potency of different surface groups in prompting complement activation in vitro and surface-mediated accumulation of inflammatory cells in vivo. Among the surfaces tested, mercaptoglycerol- and mercaptoethanol-bearing surfaces engendered the strongest inflammatory responses, as reflected by the accumulation of large numbers of adherent neutrophils and monocytes/macrophages. In contrast, L-cysteine-coated surfaces caused only minor inflammatory responses, and both glutathione-modified and untreated gold implants attracted minimal numbers of inflammatory cells. The accumulation of inflammatory cells on mercaptoglycerol surfaces appears to arise from surface-mediated complement activation because complement-depleted animals failed to exhibit inflammatory responses to mercaptoglycerol-modified implants. Furthermore, there is a close relationship between surface-mediated complement activation (as measured by in vitro iC3b/C5b-9 generation and C3 deposition) and in vivo inflammatory responses. At least in this animal model and with these model surfaces, our results indicate that surface-mediated complement activation can be responsible for the subsequent accumulation of inflammatory cells on implant surfaces.

Cellular responses to chemical and morphologic aspects of biomaterial surfaces. II. The biosynthetic and migratory response of bone cell populations

The biosynthetic and migratory response of bone cells to changes in both surface composition and morphology of polystyrene (PS) substrates was examined. A system was devised wherein micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either 0.5- or 5.0- microns-deep surface grooves. Smooth replicas (0% S) served as the control surfaces. The chemical and morphologic characteristics of the nine unique model biomaterial surfaces (MBSs) produced using this system were documented and were found to be distinct. For the biosynthetic studies, bone cells isolated from neonatal rat calvaria were plated onto the MBSs and labeled at postconfluence with [14C]proline for 24 h. Total DNA per surface, total newly synthesized collagenous (CP), and noncollagenous protein (NCP) (cell associated and secreted) were determined. Cell-associated CP was found to increase significantly for the bone cells cultured on the substrates with 0.5-micron grooves and 2% S (P < .05). Cell-associated NCP was found to be elevated for all 2% S substrates and for the 0.5-micron grooves substrates with 1% S. For the migration studies, bone cells were plated first onto 5-mm nitrocellulose disks that were attached to standard Petri dishes using a plasma clot. At confluence, the disks were removed aseptically and placed on the replicas. The cellular area occupied as a result of the outward migration of the bone cells was measured after 4 days of culture using an image analysis system. An average velocity for the leading edge of bone cell populations on each of the nine MBSs was calculated: Cells on surfaces with either 1% S or 5.0-microns grooves displayed significantly higher velocities than did the control cultures. A significant interaction effect between chemistry and morphology was observed. The biosynthetic and migratory responses of in vitro cultures of bone cells were not predictable from the observations of the cellular responses to the individual features, but appeared to depend on cellular responses to more than one substrate factor.