Shear assay measurements of cell adhesion on biomaterials surfaces

The paper examines the adhesion of human osterosarcoma (HOS) cells to selected biomaterials surfaces that are relevant to implantable biomedical systems and bio-micro-electro-mechanical systems (BioMEMS). The four biomaterials that were explored include: silicon, silicon coated with a nanoscale layer of titanium, Ti–6Al–4V, and poly-di-methy-siloxane (PDMS). The interfacial strengths between the HOS cells and the biomaterials surfaces were determined using a shear assay technique. The adhesion forces were determined using a combination of confocal microscopy images of the three-dimensional cell structure, and computational fluid dynamics (CFD) simulations that coupled actual cell morphologies and non-Newtonian fluid properties in the computation of the adhesion forces. After cell detachment by the shear assay, immunofluorescence staining of the biomedical surfaces was used to reveal the proteins associated with cell detachment. These revealed that the nano-scale Ti coating increases the cell/surface adhesion strength. Silicon with Ti coating has the strongest adhesion strength, while the other surfaces had similar adhesion strength. The measured strengths are shown to be largely associated with the detachment of focal adhesion proteins from extra-cellular matrix (ECM) proteins.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

Abstract

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Polylactide based nanostructured biomaterials and their applications

Polylactide (PLA) is one of the most innovative materials being actively investigated for a wide range of industrial applications. The polymer is a linear aliphatic thermoplastic polyester which is biodegradable as well as biocompatible, which makes it highly versatile and attractive to various commodities and medical applications. A large variety of nanoparticles of different nature and size can be blended with PLA, therefore, generating a new class of nanostructured biomaterials or nanocomposites with interesting physical properties and applications. PLA based nanostructured biomaterials are the focus of this review article, throwing light on their preparation techniques, physical properties, and industrial applications. Structural characteristics and morphological features of PLA based nanocomposites have been explained on the basis of X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Depending upon the nature and characteristics of the nanoparticles, the ultimate properties of the resulting nanocomposite materials can be tailored. Biocompatible materials such as carbon nanotubes, cellulose nanowhiskers, hydroxyapitite, etc. could be incorporated into the PLA matrix, which increase the potential of PLA for biomedical applications. Applications of PLA based nanostructured materials in different areas have been summarized.     

Protein-mediated macrophage adhesion and activation on biomaterials: a model for modulating cell behavior

The elucidation of proteins involved in biomaterial-modulated macrophage behavior is critical for the improvement of material performance and the initial exploration of material design capable of manipulating macrophage function for tissue engineering. In this paper, several in vitro and in vivo techniques are presented to demonstrate means of delineating a part of the complex molecular mechanisms involved in the interaction between biomaterial and macrophage adhesion and phenotypic development. The following conclusions were reached: (1) using radioimmunoassay, complement component C3 was found to be critical in mediating human macrophage adhesion on polyurethanes. (2) The presence of a diphenolic antioxidant additive in polyurethanes increased the propensity for complement upregulation but did not affect adherent macrophage density. (3) The subcutaneous cage-implant system was utilized to delineate interleukin-4 participation in the fusion of adherent macrophages to form foreign body giant cells in vivo in mice. The injection of purified interleukin-4 neutralizing antibody into the implanted cages significantly decreased the giant cell density; conversely, the giant cell density was significantly increased by the injection of recombinant interleukin-4 when compared with the controls. (4) The RGD and PHSRN amino acid sequences of the central cell binding domain and the PRRARV sequence of the C-terminal heparin binding domain of human plasma fibronectin were utilized to study the structure-functional relationship of protein in mediating macrophage behavior. Polyethyleneglycol-based networks grafted with the RGD-containing peptide supported higher adherent human macrophage density than surfaces grafted with other peptides. The formation of foreign body giant cell was highly dependent on the relative orientation between PHSRN and RGD domains located in a single peptide. © 1999 Kluwer Academic Publishers     

Time-dependent effects of pre-aging polymer films in cell culture medium on cell adhesion and spreading

We have tested the hypothesis that cell adhesion and spreading on polymer films are influenced by the amount of time that the polymer films are pre-aged in cell culture medium. Cell adhesion and spreading were assessed after a 6-h culture on poly(d,l-lactic acid) (PDLLA) films that had been pre-aged in cell culture medium for 30 min, 1, 3 or 7 d. Cell adhesion and spread area were enhanced as the duration of pre-aging PDLLA films in cell culture medium was increased. Materials characterization showed that the hydrophobicity and surface morphology of the PDLLA films changed with increasing length of pre-aging time. These results suggest that cell adhesion and spreading are sensitive to the time-dependent changes in PDLLA hydrophobicity and surface morphology that occur during exposure of the polymer to cell medium for different lengths of time. These results demonstrate that cell response to a degradable, biomedical polymer can change as a function of the amount of time that the polymer is exposed to physiological medium. This article, a contribution of the National Institute of Standards and Technology, is not subject to US copyright.     

Controlling pre-osteoblastic cell adhesion and spreading on glycopolymer brushes of variable film thickness

Controlling the cell behavior on biocompatible polymer surfaces is critical for the development of suitable medical implant coatings as well as in anti-adhesive applications. Synthetic glycopolymer brushes, based on sugar methacrylate monomers have been reported as robust surfaces to resist protein adsorption and cell adhesion. In this study, poly(D-gluconamidoethyl methacrylate) (PGAMA) brushes of various chain lengths were synthesized directly from initiator functionalized glass substrates using surface-initiated atom transfer radical polymerization. The glycopolymer film thicknesses were determined by ellipsometry, whereas the wettability and the morphology of the surfaces were characterized by static water contact angle measurements and atomic force microscopy, respectively. Stable, grafted films with thicknesses in the dry state between 4 and 20?nm and of low roughness (~1?nm) were obtained by varying the polymerization time. Cell experiments with MC3T3-E1 pre-osteoblasts cultured on the PGAMA brushes were performed to examine the effect of film thickness on the cell morphology, cytoskeleton organization and growth. The results revealed good cell spreading and proliferation on PGAMA layers of low film thickness, whereas cell adhesion was prevented on polymer films with thickness higher than ~10?nm, indicating their potential use in medical implants and anti-adhesive surfaces, respectively.

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Mesenchymal stem cellular adhesion and cytotoxicity study of random biopolyester scaffolds for tissue engineering

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rabbits and inoculated respectively on 3D scaffolds of poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV), poly(butylenes succinate) (PBS) and different blends (100/0, 80/20, 50/50, 20/80, 0/100) (Wt%) in vitro. It was found that the (50/50) blends possessed the best performance on adhesion and cytotoxicity of MSCs. The scanning electronic microscopy (SEM) results showed that the (50/50) blends had the appropriate roughness for MSCs to attach and grow, which may be used as a suitable biomaterial to create small caliber vascular grafts.     

A comparison of different nanostructured biomaterials in subcutaneous tissue

The nanostructured surface of a material can improve its interaction with cells and its acceptance as an implant. We compared two novel biomaterials with different nanostructures: Bioverit((R)) II with a coating of nanoporous silica and chitosan-hydroxyapatite composite materials. Pure Bioverit((R)) II served as a control. Platelets of these materials were implanted for 28, 85 and 300 days in the subcutaneous tissue in the neck of 38 rabbits. After excising the specimens they were fixed, embedded in epoxy resin and analyzed histologically. All coated Bioverit((R)) II implants showed a thin capsule of connective tissue. After 300 days, these capsules tended to be thicker than in pure Bioverit((R)) II. No signs of inflammation were observed and the materials appeared unaltered by visual inspection. In case of chitosan-hydroxyapatite composites, massive capsules consisting of dense connective tissue were found, and the material showed signs of biodegradation in form of fissures and cavities. In conclusion, the nanoporous coating showed no obvious positive effect with regard to capsule formation; the chitosan-hydroxyapatite implants provoked a stronger interaction between cells and material. However, most Bioverit((R)) II implants showed no alterations optically, whereas chitosan-hydroxyapatite was partly degraded in all cases.     

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both α5 and αv-containing integrin heterodimers, whereas only αv integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of α2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Plasma- and chemical-induced graft polymerization on the surface of starch-based biomaterials aimed at improving cell adhesion and proliferation

Plasma and chemical induced graft polymerization of acrylic monomers on starch-based biomaterials has been performed with the aim to improve cell adhesion and proliferation on the surface of the polymers, in order to adequate their properties for bone tissue engineering scaffolding applications. Plasma and chemical surface activation was aimed to induce the polymerization of acrylic polar monomers being carried out by applying a radio frequency plasma and expose the samples to a mixture of Ar/O₂, or by immersion in a H₂O₂/(NH₄)₂S₂O₈ solution with UV radiation, respectively. Both procedures were followed by the graft polymerization of the corresponding monomers. Polymer grafting was analyzed by Fourier transformed infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and by contact angle measurements. Properties such as mechanical performance, swelling degree, and degradation behavior, as well as bioactivity, have been studied and compared for the different activation methods. Finally, preliminary cell adhesion and proliferation tests were performed, using goat bone marrow cells, showing a remarkable improvement with respect to original non-surface modified starch-based biomaterials.     

Reduction of ice adhesion on nanostructured and nanoscale slippery surfaces

Ice nucleation and accretion on structural surfaces are sources of major safety and operational concerns in many industries including aviation and renewable energy. Common methods for tackling these are active ones such as heating, ultrasound, and chemicals or passive ones such as surface coatings. In this study, we explored the ice adhesion properties of slippery coated substrates by measuring the shear forces required to remove a glaze ice block on the coated substrates. Among the studied nano...     

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.     

Phosphate and cell growth on nanostructured semiconductors

Abstracts are not published in this journal This revised version was published online in November 2006 with corrections to the Cover Date.     

Preliminary study on human protein adsorption and leukocyte adhesion to starch-based biomaterials

In this study, the adsorption of human serum albumin (HSA), fibronectin (FN) and vitronectin (VN) onto the surface of novel biodegradable materials was evaluated by immunostaining. Specifically, polymeric blends of corn starch with cellulose acetate (SCA), ethylene vinyl alcohol copolymer (SEVA-C), and polycaprolactone (SPCL) were immersed in unitary and competitive systems; that is, binary and more complex protein solutions. For binary solutions, different HSA and FN protein distribution patterns were observed depending on the starch-based surface. Furthermore, the relative amount of proteins adsorbed onto starch-based surfaces was clearly affected by protein type: a preferential adsorption of VN and FN as compared to HSA was observed. On tests carried out with unitary, binary and more complex solutions, it was found that vitronectin adsorption ability was enhanced in competitive systems, which was associated with a lower amount of adsorbed albumin. In order to assess the effect of these human proteins on cell behavior, a mixed population of human lymphocytes and monocytes/macrophages was cultured over pre-coated SEVA-C surfaces. Through anti-CD3 and CD-14 monoclonal antibody labeling and cell counting, leukocyte adhesion onto pre-coated SEVA-C surfaces was analyzed. Based on the results, it was possible to detect albumin long-term effects and fibronectin short-term effects on cell adhesion proving that previously adsorbed proteins modulate leukocyte behavior.     

Multilayer coatings on biomaterials for control of MG-63 osteoblast adhesion and growth

Here, the layer-by-layer technique (LbL) was used to modify glass as model biomaterial with multilayers of chitosan and heparin to control the interaction with MG-63 osteoblast-like cells. Different pH values during multilayer formation were applied to control their physico-chemical properties. In the absence of adhesive proteins like plasma fibronectin (pFN) both plain layers were rather cytophobic. Hence, the preadsorption of pFN was used to enhance cell adhesion which was strongly dependent on pH. Comparing the adhesion promoting effects of pFN with an engineered repeat of the FN III fragment and collagen I which both lack a heparin binding domain it was found that multilayers could bind pFN specifically because only this protein was capable of promoting cell adhesion. Multilayer surfaces that inhibited MG-63 adhesion did also cause a decreased cell growth in the presence of serum, while an enhanced adhesion of cells was connected to an improved cell growth.     

Interactions between neural stem cells and biomaterials combined with biomolecules

Neural repair and regeneration have been a tough problem in clinical studies. Tissue engineering using biomaterials along with neural stem cells (NSCs) have shown great potential for treatment, especially along with the biomolecules to regulate the NSCs can get more promising results. The biomolecules in the materials have a favorable impact on cell adhes ion, expansion, and differentiation. Thus, the interactions between biomaterials loading biomolecules and NSCs also receive particular attention. In this review, recent progresses of modified biomaterials by such biomolecules for neural injury and their impact on NSCs behavior will be discussed.     

Designing nanostructured block copolymer surfaces to control protein adhesion

The profile and conformation of proteins that are adsorbed onto a polymeric biomaterial surface have a profound effect on its in vivo performance. Cells and tissue recognize the protein layer rather than directly interact with the surface. The chemistry and morphology of a polymer surface will govern the protein behaviour. So, by controlling the polymer surface, the biocompatibility can be regulated. Nanoscale surface features are known to affect the protein behaviour, and in this overview the nanostructure of self-assembled block copolymers will be harnessed to control protein behaviour. The nanostructure of a block copolymer can be controlled by manipulating the chemistry and arrangement of the blocks. Random, A-B and A-B-A block copolymers composed of methyl methacrylate copolymerized with either acrylic acid or 2-hydroxyethyl methacrylate will be explored. Using atomic force microscopy (AFM), the surface morphology of these block copolymers will be characterized. Further, AFM tips functionalized with proteins will measure the adhesion of that particular protein to polymer surfaces. In this manner, the influence of block copolymer morphology on protein adhesion can be measured. AFM tips functionalized with antibodies to fibronectin will determine how the surfaces will affect the conformation of fibronectin, an important parameter in evaluating surface biocompatibility.