George Altankov

Reorganization of substratum-bound fibronectin on hydrophilic and hydrophobic materials is related to biocompatibility

It is a general trend that mammalian cells interact better with wettable surfaces than with non-wettable surfaces. The basis for this difference is still poorly understood. In this study hydrophilic clean glass and hydrophobic octadecyl glass have been used as model surfaces. We show that fibroblasts on hydrophilic surfaces may reorganize fluorescent fibronectin (FN) in an extracellular matrix-like structure whereas on hydrophobic surfaces no rearrangement of FN occurs. This was accompanied by a high proliferation of fibroblasts on clean glass whereas on octadecyl glass no cell growth occurred. Moreover, it was demonstrated that there are striking differences in the morphology of fibroblasts adhering to hydrophilic and hydrophobic surfaces, judged by the overall cell shape, the organization of FN receptors and actin filaments. Indeed, the preadsorption of FN on these surfaces could almost abolish morphological differences between hydrophilic and hydrophobic surfaces. However, preadsorption of FN could not restore the proliferation of fibroblasts on the hydrophobic surface. Taken together, the results suggest that the method of adsorption and reorganization of FN may be critical for the biocompatibility of materials.

Studies on cell-biomaterial interaction: role of tyrosine phosphorylation during fibroblast spreading on surfaces varying in wettability

In a previous study we observed that protein tyrosine phosphorylation was significantly diminished in the focal adhesions of human fibroblasts attached on a hydrophobic surface in comparison with hydrophilic glass. This result raises the possibility that the tyrosine phosphorylation pathway may be involved in the regulation of cell-biomaterial interaction. To learn more about the interaction of anchorage-dependent cells with biomaterials, four different materials with wettability ranging from hydrophilic (water contact angle 25 degrees) to hydrophobic (water contact angle 111 degrees) were investigated, i.e. clean glass (glass), aminopropylsilane (APS), octadecylsilane (ODS) and silicone (SI). Immunofluorescence microscopy revealed increased stress formation and fibronectin (FN) receptor-rich focal adhesions for fibroblasts attached on more hydrophilic surfaces (glass and APS) in comparison to the relatively hydrophobic materials (ODS and SI). Phosphorylation of tyrosine residues, also studied by immunofluorescence microscopy, was considerably higher on glass and APS, lower for ODS, negligible for SI, and was found to colocalize with FN receptor-rich focal adhesions. Preadsorption of FN tended to restore cell adhesion and spreading on the hydrophobic ODS and SI. Quantitative data on cell proliferation and tyrosine phosphorylation showed moderate wettable material maximum values for APS, followed by glass. ODS and SI, demonstrating a non-linearity of these parameters with the wettability of materials. Interestingly, the preadsorption of FN increased both parameters, particularly for the hydrophobic materials ODS and SI. Phosphorylation of tyrosine on FN-coated substrata was corroborated by the accessibility of binding sites estimated by ELISA using polyclonal and monoclonal FN antibodies. Our results suggest that measurement of the phosphotyrosine activity of cells may be a sensitive parameter for the ability of biomaterials to support the attachment and proliferation of cells.

Preparation of PLLA/PEG nanofibers by electrospinning and potential applications

Poly(L-lactide) (PLLA)/polyethylene glycol (PEG) mixed solutions were successfully electrospun into micro-or nanofibrous polymer mats. The fiber diameter was in the range 100nm-6μm. The effect of the concentration of the spinning solutions and the ratio of PLLA/PEG on the fiber diameter and morphology was investigated. The hydrophilicity was tuned by varying the PLLA/PEG ratio. The tissue compatibility of the electrospun nanofibrous scaffolds was screened using two different cell models of human dermal fibroblasts and the osteoblast-like cell line MG-63. Both types of cells attached uniformly and approximately equally to all PLLA/PEG nanofibers. In long-term cultures osteoblast-like cells tend to spatially organize in tissue-like structure, particularly within the scaffold with the highest PEG content (PLLA/PEG at weight ratio 70/30). These results indicate that PLLA mixed with hydrophilic PEG produces a promising new biocompatible material for engineering scaffolds.

Membranes for biohybrid liver support systems--investigations on hepatocyte attachment, morphology and growth.

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.

Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion

Hydroxyapatite and hybrid gelatine/hydroxyapatite microspheres were obtained through a water in oil emulsion of a calcium phosphate cement (CPC). The setting reaction of the CPC, in this case the hydrolysis of α-tricalcium phosphate, was responsible for the consolidation of the microspheres. After the setting reaction, the microspheres consisted of an entangled network of hydroxyapatite crystals, with a high porosity and pore sizes ranging between 0.5 and 5 μm. The size of the microspheres was tailored by controlling the viscosity of the hydrophobic phase, the rotation speed, and the initial powder size of the CPC. The incorporation of gelatin increased the sphericity of the microspheres, as well as their size and size dispersion. To assess the feasibility of using the microspheres as cell microcarriers, Saos-2 cells were cultured on the microspheres. Fluorescent staining, SEM studies, and LDH quantification showed that the microspheres were able to sustain cell growth. Cell adhesion and proliferation was significantly improved in the hybrid gelatin/hydroxyapatite microspheres as compared to the hydroxyapatite ones.

Fibronectin activity on substrates with controlled OH density

Adhesion of human fibroblast to a family of fibronectin (FN) coated model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios to obtain a controlled surface density of --OH groups was investigated. Cell adhesion and spreading surprisingly decreased as the fraction of --OH groups on the surface increased. AFM studies of FN conformation revealed formation of a protein network on the more hydrophobic surfaces. The density of this network diminished as the fraction of --OH groups in the sample increased, up to a maximal --OH concentration at which, instead of the network, only FN aggregates were observed. The kinetics of network development was followed at different adsorption times. Immunofluorescence for vinculin revealed the formation of well-developed focal adhesion complexes on the more hydrophobic surface (similar to the control glass), which became less defined as the fraction of --OH groups increased. Thus, the efficiency of cell adhesion is enhanced by the formation of FN networks on the substrate, directly revealing the importance of the adsorbed protein conformation for cell adhesion. However, cell-dependent reorganization of substrate-associated FN, which usually takes place on more hydrophilic substrates (as do at the control glass slides), was not observed in this system, suggesting the increased strength of protein-to-substrate interaction. Instead, the late FN matrix formation--after 3 days of culture--was again better pronounced on the more hydrophobic substrates and decreased as the fraction of --OH groups increase, which is in a good agreement with the results for overall cell morphology and focal adhesion formation.

Discerning the Role of Topography and Ion Exchange in Cell Response of Bioactive Tissue Engineering Scaffolds

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

Remodeling of fibrinogen by endothelial cells in dependence on fibronectin matrix assembly. Effect of substratum wettability

The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as fibronectin (FN) and fibrinogen (FNG). In this study, we show that the deposition of fibrinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. β3 integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG fibrillogenesis required the activity of the β1 integrin. Studies of EC morphology showed the predominant peripheral organization of actin filaments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed fibrinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in fibrilar structures.

Biological Activity of the Substrate-Induced Fibronectin Network: Insight into the Third Dimension through Electrospun Fibers

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.

Fibronectin matrix formation and the biocompatibility of materials

The formation of an extracellular matrix (ECM) was investigated by the secretion of cellular FN during 72 h incubation of fibroblasts on defined hydrophilic glass and hydrophobic octadecyl glass (ODS). It was found, that the ability of fibroblasts to form their own ECM was inhibited on the hydrophobic ODS in comparison to glass, where significant amounts of FN were deposited in fibrils and clusters. This result was corroborated by the impaired morphology of cells on ODS, visualized by staining of actin micro-filaments and the FN receptor. Moreover, it was found that cell growth was significantly inhibited on the hydrophobic surface. In contrast to these findings, cell morphology and proliferation was not impaired on glass. Precoating of both substrata with FN could restore the cell morphology and enhanced the proliferation on the hydrophobic ODS. ELISA for FN binding using polyclonal and monoclonal antibodies revealed that under these circumstances total FN adsorption, as well as the presence of cell- and heparin-binding domains was much higher on ODS in comparison to glass.