Adhesion and growth of CaCo2 cells on surface-modified PEEK substrata

A series of surface-functionalized poly(ether ether ketone) (PEEK) films has been prepared by selective wet-chemistry; they are hydroxylated polymer (PEEK-OH) obtained by reduction, aminated polymer (PEEK-[]-NH2) prepared by coupling a diisocyanate reagent to PEEK-OH (PEEK-[]-NCO) followed by hydrolysis, and carboxylated and aminocarboxylated polymers (PEEK-[]-GABA and PEEK-Lysine) resulting from the coupling of aminoacids to PEEK-[]-NCO. The aminated and carboxylated substrata promoted the adhesion and growth of CaCo2 cells in the presence of serum. Fibronectin (FN), an extra-cellular matrix protein, has been covalently fixed and/or adsorbed on various PEEK substrata, in the presence or not of a polymeric surfactant (Pluronic F68). The performances of the FN-grafted substrata (PEEK-[]-FN(1) and PEEK-[]-FN(2)) were significantly higher than those of reference substrata simply coated with FN (PEEK-OH(+FN)(1) and (2), PEEK-[]-NH2(+FN)(1) and (2)), considering the adhesion and spreading of CaCo2 cells in the absence of serum. Moreover, the stability of the adherent cells on the FN-adsorbed substrata dramatically depended on the experimental conditions applied during the PEEK coating with FN.     

Fibronectin adsorption or/and covalent grafting on chemically modified PEEK film surfaces.

Poly(ether ether ketone) (PEEK) films were chemically modified, by surface wet chemistry, into PEEK-OH, PEEK-NH2, and PEEK-NCO. Fibronectin (FN) adsorption, in the presence or absence of two non-ionic surfactants, was compared onto PEEK, PEEK-OH, and PEEK-NH2 on which the protein can only be adsorbed, and onto PEEK-NCO on which FN could be covalently grafted. The amounts of FN present on the various supports were assayed by ELISA and LSC (with 125I-labeled FN). The remarkable effect of Pluronic F68 in preventing non-specific protein adhesion on the less hydrophilic surfaces was pointed out. Accordingly, a procedure could be proposed that allows minimal FN adhesion vs FN fixation on PEEK-NCO. The resulting PEEK-FN film, which immobilized 120-150 ng FN cm(-2), constitutes a new substratum for cell cultivation.     

Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion.

Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence. Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane. The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure. The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates. However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3. The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN.     

Morphological evidence for a different fibronectin receptor organization and function during fibroblast adhesion on hydrophilic and hydrophobic glass substrata

A polyclonal antibody against the β1 subunit of the fibronectin (FN) receptor was used to mimic the early events of integrin receptor functioning to study the initial cellular processes during the organization of FN matrix on biomaterials. Hydrophilic glass and hydrophobic octadecylsilane (ODS) surfaces have been applied as models for different biocompatible materials. By immunofluorescence we could demonstrate that FN receptors organize on the dorsal cell surface of adhering fibroblasts in a specific linear pattern along with actin filaments, but only if the cells were attached to hydrophilic glass. In contrast, FN receptors were not reorganized on hydrophobic octadecylsilane (ODS). In parallel experiments, FN matrix formation after 72 h of incubation on the same substrata has been analyzed microscopically, and quantified by cell ELISA, in order to be further correlated with the integrin receptor functioning in contact with the biomaterials. It was found that FN structuring and the amount of FN matrix have been significantly diminished on ODS that was related to the observed changes in integrin receptor functioning. To learn more about the mechanism of this phenomenon, desorption of 125-FN from these substrata was studied and found to be significantly decreased on hydrophobic ODS. As a consequence, FN receptor (function) might be arrested on the ventral cell surface, thus the important role of β 1 integrins in the positional organization of the FN matrix may be disturbed. In light of these facts, antibody-induced clustering of FN receptor can be considered as a useful model for studying the early steps of FN matrix formation on biomaterials.     

Modulating the biocompatibility of polymer surfaces with poly(ethylene glycol): effect of fibronectin

A novel approach described earlier for improving polymer substratum biocompatibility(1) is further elucidated. Polysulfone (PSf) spin-coating films were modified by covalent end-on grafting of hydrophilic and sterically demanding photo-reactive poly(ethylene glycol) (PEG) conjugates (ABMPEG; 10 kDa). The degree of grafting density was varied systematically, yielding a wide spectrum of attained surface characteristics monitored by air-water contact angles (captive bubble method). Fibronectin (FN) adsorption was studied by in situ ellipsometry and found to decrease monotonically as ABMPEG grafting density increased. The adhesive interaction of human skin fibroblasts with these substrata and, in particular, the effect of FN precoating were investigated in detail. A clear optimum of cell-substratum interactions was found for mildly modified substrata, employing well established microscopic and immunofluorescence techniques, namely the monitoring of cell adhesion and spreading, overall cell morphology, organization of FN receptors, and focal adhesions as well as FN matrix formation. The results suggest that cell interactions with hydrophobic polymer substrata are enhanced considerably when modified with hydrophilic and sterically demanding PEG moieties at a low surface coverage due to enhanced biologic activity of adsorbed and intercalated adhesive proteins such as FN.     

Fabrication and enzymatic degradation of fibronectin-based ultrathin films

Novel fibronectin (FN)-based ultrathin films were prepared by layer-by-layer (LbL) assembly. Among the various combinations of extracellular matrix (ECM) proteins and glycosaminoglycans (GAGs) such as FN, gelatin (G), α-elastin (E) and heparin (Hep), FN/Hep, FN/G and FN/E nanofilms were successfully fabricated in phosphate buffer solutions (pH 7.4). The film thickness of the nanofilms, in which each component can interact with each other by FN-specific interactions, was larger than that of other LbL films (E/Hep, G/E and G/Hep) prepared by electrostatic interactions. The FN/G film was rapidly decomposed by treatment with elastase, thus demonstrating, the enzymatic biodegradability of the nanofilm. We prepared the FN/heparinoid multilayers composed of FN and dextran sulfate (Dex), and its thickness was much larger than that of the FN/α-poly(L-lysine hydrochloride) (PLL) film prepared by LbL assembly using common electrostatic interactions. Furthermore, the FN/G and FN/Dex nanofilms prepared by FN-specific interaction were more stable in Eagle's MEM with 10% fetal bovine serum (FBS) than the electrostatic assembling films, FN/PLL and PLL/Dex. FN-based multilayers composed of FN and ECM components can be useful as artificial ECM films for tissue engineering and other biomedical applications.     

Adhesion of Staphylococcus epidermidis to biomaterials is inhibited by fibronectin and albumin

Decades of contradictory results have obscured the exact role of adsorbed fibronectin in the adhesion of the bacterium, Staphylococcus epidermidis, to biomaterials. Here, the ability of adsorbed fibronectin (FN) or bovine serum albumin (BSA) to modulate S. epidermidis adhesion to various biomaterials is reported. FN or BSA was adsorbed in increasing surface densities up to saturated monolayer coverage onto various common biomaterials, including poly(ethylene terephthalate), fluorinated ethylene propylene, poly(ether urethane), silicone, and borosilicate glass. Despite the wide range of surface characteristics represented, adsorption isotherms varied only subtly between materials for the two proteins considered. S. epidermidis adhesion to the various protein-coated biomaterials was quantified in a static-fluid batch adhesion assay. Although slight differences in overall adherent cell numbers were observed between the various protein-coated substrata, all materials exhibited significant dose-dependent decreases in S. epidermidis adhesion with increasing adsorption of either protein (FN, BSA) to all surfaces. Results here indicate that S. epidermidis adhesion to FN-coated surfaces is not a specific adhesion (i.e., receptor: ligand) mediated process, as no significant difference in adhesion was found between FN- and BSA-coated materials. Rather, results indicate that increasing surface density of either FN or BSA actually inhibited S. epidermidis adhesion to all biomaterials examined.     

Effects of controlled fibronectin surface orientation on subsequent Staphylococcus epidermidis adhesion

Several bacterial species, including Staphylococcus aureus and Staphylococcus epidermidis (SE) are known to express cell receptors that bind specifically to surface immobilized or extracellular matrix ligands, such as the protein fibronectin (FN). Yet, few existing studies have examined the effect of protein surface orientation on bacterial adhesion. We report here a substratum modification protocol that allows for the specific orientation of FN molecules on a surface at known levels of surface coverage. Monoclonal antibodies (Mabs), specific to either the COOH-terminus or NH3-terminus of FN, are conjugated to biotin, then immobilized to streptavidin-coated glass substrata. Specific orientation of the bound FN molecules is verified using the same Mabs in an ELISA. Bacterial adhesion of Staphylococcus epidermidis (SE) to FN bound by either its C-terminus or its NH3-terminus was quantified in batch static adhesion assays. Results indicate an increase in SE adhesion to FN-coated surfaces when the FN is bound by its C-terminus (NH3-terminus free), indicating SE receptor-specific adhesion to the FN NH3-terminus. These studies demonstrate that antifibronectin monoclonal antibodies can be used to specifically bind and orient fibronectin on a surface. In addition, adhesion of SE to these model substrata can be controlled by the orientation of the protein.     

Cell adhesive PET membranes by surface grafting of RGD peptidomimetics

A non-peptide mimic of the Arg-Gly Asp (RGD) active sequence of adhesive proteins (such as vitronectin) has been equipped with two different spacer-arms for surface anchorage. The covalent grafting on poly(ethylene terephthalate) (PET) membrane was realized via the activation of the hydroxyl polymer chain-ends by tosylation followed by nucleophilic substitution. The surface density of peptidomimetics was determined by X-ray photoelectron spectroscopy (XPS), on the basis of F/C atomic ratios since a fluorine tag was incorporated into the RGD-like compounds. The biological activity of soluble peptidomimetics was evaluated versus isolated human integrin alpha(v)beta(3) (vitronectin receptor), and versus CaCo2 cells. Inhibition of cellular adhesion was observed after pre-incubation of CaCo2 cells with soluble peptidomimetics. On the other hand a significant promotion of cellular adhesion resulted from the surface grafting of peptidomimetics on the PET culture substrate. The best performance was obtained with the RGD-like integrin ligand bearing a triethylene glycol spacer-arm.     

Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells

Interaction between hematopoietic stem/progenitor cells (HSPCs) and their extra cellular matrix components is an integral part of the signaling control for HSPC survival, proliferation and differentiation. We hypothesized that both substrate topographical cues and biochemical cues could act synergistically with cytokine supplementation to improve ex vivo expansion of HSPCs. In this study, we compared the ex vivo expansion of human umbilical cord blood CD34⁺ cells on unmodified, hydroxylated, carboxylated and aminated nanofibers and films. Results from 10-day expansion cultures showed that aminated nanofiber mesh and film were most efficient in supporting the expansion of the CD34⁺CD45⁺ cells (195-fold and 178-fold, respectively), as compared to tissue culture polystyrene (50-fold, p<0.05). In particular, aminated nanofiber meshes supported a higher degree of cell adhesion and percentage of HSPCs, as compared to aminated films. SEM imaging revealed the discrete colonies of cells proliferating and interacting with the aminated nanofibers. This study highlights the potential of a biomaterials approach to influence the proliferation and differentiation of HSPCs ex vivo.     

Adsorption characteristics of human plasma fibronectin in relationship to cell adhesion

Adsorption of human plasma fibronectin (FN) on nonsulfonated and sulfonated polymer surfaces was studied, by using a polyclonal antiserum to FN and the ELISA method. ELISA signal was recorded as a function of FN concentration in solutions. The concentration dependence of FN binding shows the saturation effect in the range 5-10 microg/mL. ELISA data are discussed in the terms of a self-assembled monolayer and different conformations of the FN molecule.The early adhesion of L1210 cells to polymer surfaces after prior adsorption of FN on these surfaces was studied under static conditions. In the case of FN adsorbed on sulfonated surfaces, the relative number of adhering cells increased with the increase of the interfacial surface tension (i.e., the cell adhesion depends on the surface density of sulfonic groups). However, in the case of FN adsorbed on nonsulfonated surfaces, the relative number of adhering cells was low and independent on the interfacial surface tension.The alpha(5)beta(1)-integrin blocking by a monoclonal antibody resulted in a strong inhibition of the cell adhesion to FN adsorbed on sulfonated polymer surfaces. This indicates that cell adhesion to FN adsorbed on these surfaces is mostly mediated by the alpha(5)beta(1)-integrin. In contrast, in the case of FN adsorbed on nonsulfonated surfaces the cell adhesion was not inhibited by the alpha(5)beta(1)-integrin blocking.     

聚合物表面生物修饰对肌腱细胞黏附特性的影响

为了探讨增强肌腱细胞与聚合物材料黏附力学特性的措施 ,采用生物可降解聚合物—乳酸与羟基乙酸共聚物 85 / 15 ,制成透光的薄膜 ,在膜表面裱衬聚赖氨酸的基础上 ,表面裱衬细胞外基质 ( I型胶原蛋白、纤维粘连蛋白 ,以及相应的抗体 )和生长因子 (类胰岛素生长因子 1) ,接种转化人胚肌腱细胞后 ,利用微吸管实验技术测定转化人胚肌腱细胞与聚合物薄膜的黏附力。结果显示 :在聚合物薄膜表面裱衬纤维粘连蛋白或 I型胶原蛋白 ,可明显提高转化人胚肌腱细胞与聚合物薄膜的黏附力 ( P<0 .0 5 ) ,但若在此基础上进一步分别复合裱衬纤维粘连蛋白抗体或 I型胶原蛋白抗体 ,则引起转化人胚肌腱细胞与聚合物薄膜的黏附力明显下降 ( P<0 .0 5 ) ;肌腱细胞对聚合物薄膜的黏附力与细胞外基质蛋白 (纤维粘连蛋白或 I型胶原蛋白 )的裱衬浓度有很好的依赖性 ;I型胶原蛋白和纤维粘连蛋白介导转化人胚肌腱细胞与聚合物薄膜的特异性黏附作用 ;二者复合裱衬浓度达到一定比例时 ,可产生协同作用 ,增强黏附效果 ;这种特异性黏附作用可被相应的抗体分子所抑制 ;生长因子对转化人胚肌腱细胞有明显的促黏附作用。提示 ,材料表面生物修饰可促进转化人胚肌腱细胞与聚合物的黏附作用 ,这对构建组织工程化肌腱具有重要的指导意义     

Surface modification of poly(ether ether ketone) with methacryloyl-functionalized phospholipid polymers via self-initiation graft polymerization

To improve blood compatibility of poly(ether ether ketone) (PEEK), surface modification with methacryloyl-functionalized phospholipid polymers was performed through self-initiation graft polymerization. The copolymers (PMA) of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-aminoethyl methacrylate hydrochloride were synthesized by conventional free radical polymerization. The PMA was then immobilized with pentafluorophenyl methacrylate to obtain methacryloyl-functionalized MPC polymers (PMAMA). The degree of substitution of the methacryloyl group into the copolymer was nearly completed. The PMAMA was dissolved in 1-butanol and the solution was dropped on PEEK. UV light (350?±?50?nm) was subsequently irradiated on PEEK for various periods. Elemental analysis of the PEEK surface was performed by X-ray photoelectron spectroscopy and phosphorus and nitrogen signals due to the MPC units on PEEK were observed. The surface wettability of PEEK was also improved by immobilization of PMAMA. Plasma protein adsorption was effectively reduced on the PMAMA-immobilized surface regardless of the type of protein. Furthermore, PMAMA immobilization was also useful in reducing platelet adhesion on PEEK. In conclusion, methacryloyl-functionalized MPC polymers could be immobilized on PEEK by simple photo-irradiation, resulting in significant improvement in blood compatibility.     

Preparation,mechanical properties and in vitro cytocompatibility of multi-walled carbon nanotubes/poly(etheretherketone) nanocomposites

Desired bone repair material must have excellent biocompatibility and high bioactivity. Moreover, mechanical properties of biomaterial should be equivalent to those of human bones. For developing an alternative biocomposite for load-bearing orthopedic application, combination of bioactive fillers with polymer matrix is a feasible approach. In this study, a series of multi-walled carbon nanotubes (MWCNTs)/poly(etheretherketone) (PEEK) bioactive nanocomposites were prepared by a novel coprecipitation-compounding and injection-molding process. Scanning electron microscope (SEM) images revealed that MWCNTs were adsorbed on the surface of PEEK particles during the coprecipitation-compounding process and dispersed homogeneously in the nanocomposite because the conjugated PEEK polymers stabilized MWCNTs by forming strong π–π stack interactions. The mechanical testing revealed that mechanical performance of PEEK was significantly improved by adding MWCNTs (2–8 wt%) and the experimental values obtained were close to or higher than that of human cortical bone. In addition, incorporation of MWCNTs into PEEK matrix also enhanced the roughness and hydrophilicity of the nanocomposite surface. In vitro cytocompatibility tests demonstrated that the MWCNTs/PEEK nanocomposite was in favor of cell adhesion and proliferation of MC3T3-E1 osteoblast cells, exhibiting excellent cytocompatibility and biocompatibility. Thus, this MWCNTs/PEEK nanocomposite may be used as a promising bone repair material in orthopedic implants application.     

Effect of micro-roughening of poly(ether ether ketone) on bone marrow derived stem cell and macrophage responses,and osseointegration

Poly(ether ether ketone) (PEEK) has emerged as a candidate to replace metal implants because of its satisfactory mechanical properties, radiolucency, and lack of metal allergy. However, PEEK lacks osseointegration ability limiting its clinical applications. To overcome this problem, we prepared PEEK with a micro-rough surface using the sandblast method to modulate its osseointegration property; the sandblast method is simple, cost-effective, and is already applied to clinical metal implants. The surface roughness of the sandblasted PEEK was about 2.3 μm, whereas that of mirror-polished PEEK was 0.06 μm. Rat bone marrow-derived mesenchymal stem cells (RMSCs) showed higher proliferation, osteocalcin (OC) expression and bone-like nodule formation on micro-roughened PEEK compared with those cultured on mirror-polished PEEK, suggesting that micro-roughening facilitated RMSCs proliferation and differentiation. The micro-roughened surface slightly mitigated secretion of inflammatory C-C motif chemokine 2 (CCL-2) from lipopolysaccharide (LPS)-stimulated macrophages, but not of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). Finally, to compare osseointegration, specimens were implanted in rat femur bone marrow cavities, and then the pull-out force was measured. The pull-out force of micro-roughened PEEK was about four times higher than that of the mirror-polished PEEK. These results showed that micro-roughening of PEEK using the sandblast method was able to improve osseointegration, partly through elevating proliferation and differentiation of RMSCs.     

Type I and type IV collagen promote adherence and spreading of human type II pneumocytes in vitro

Human lung tissue was enzymatically digested with dispase and type II pneumocytes were subsequently separated on a discontinuous metrizamide gradient. In primary adherence assays with freshly isolated cells, mean adherence values of 21.7, 19.6, 13.4, 6.8 and 7.0% were obtained after 48 hours on type I collagen (CI), type IV collagen (CIV), fibronectin (FN), laminin (LM) and tissue culture plastic, respectively. Secondly, readherence and spreading assays were performed with type II pneumocytes, previously cultured on CI, FN, matrix of murine EHS cells, and culture plastic. After a culture period of 48 to 72 hours, the cells were replated and tested on CI, CIV, FN, LM, CI + FN, CI + LM, CIV + FN and CIV + LM substrata in order to find out whether preculture substrata can modulate the adherence response of these cells. Readherence values, 1 hour after replating, were 3.3- to 8.6-fold higher than primary adherence values after 48 hours. The highest readherence values were always obtained on collagen-containing substrata after preculture on fibronectin. Spreading values after replating ranged between 35.4 and 6.6% on collagen-containing substrata, the highest values were again obtained after preculture on FN. Less than 6.9% and 0.5% of the cells spread on pure FN and pure LM, respectively. The data of the present investigation indicate that human type II pneumocytes adhere and spread preferentially on collagenous substrata rather than on other components of the extracellular matrix. This might be important in helping us to understand the functional in vivo activity of these cells, especially as regards re-epithelialization of the injured pulmonary alveolus.     

Polymeric coatings that mimic the endothelium: combining nitric oxide release with surface-bound active thrombomodulin and heparin

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N(2)O(2))). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.     

Fibronectin anchorage to polymer substrates controls the initial phase of endothelial cell adhesion

Early stages of the adhesion of human endothelial cells onto a set of smooth polymer films were analyzed to reveal the modulation of cell-matrix interactions by the physicochemical constraints of predeposited fibronectin (FN). Hydrophobic and hydrophilic polymer substrates, consisting of poly(octadecene-alt-maleic anhydride) and poly(propene-alt-maleic anhydride) films, were coated with similar amounts of FN at conditions of either covalent or noncovalent immobilization. The well-defined substrates permit variation of the anchorage of FN at invariant topography, pliability, and molecular composition. Although all of the compared FN coatings were effective in stimulating attachment of endothelial cells, the initial formation of cell-matrix adhesions was found to be controlled by the type of interaction between predeposited FN and the underlying substrate. Covalent linkage and hydrophobic interactions of the predeposited FN with the polymer films interfered with the rapid generation of focal and fibrillar adhesions. It was demonstrated that this was caused by the fact that only weakly bound FN could become readily reorganized by the adherent cells. Upon prolonged culture periods at standard cell culture conditions, secretion and deposition of organized extracellular matrix by the attached cells was found to balance out the differences of the substrates.     

Semiquantitative evaluation of fibronectin adsorption on unmodified and sulfonated polystyrene, as related to cell adhesion

The process of human fibronectin (FN) adsorption on nonsulfonated and sulfonated polystyrene surfaces was studied in relation to mechanisms of L1210 cell adhesion. Radioisotope assays directed towards FN, as well as ELISA measurements of adsorbed FN and bovine serum albumin (BSA) were carried out. (125)I radioisotope assays led to linear FN adsorption isotherms. When combined to ELISA measurements for FN, they revealed the multilayer adsorption. Results indicated a large difference in the saturating first-layer surface density of FN adsorbed on sulfonated and nonsulfonated polystyrene surfaces: significantly (ca. factor of 5) less FN molecules are necessary to complete a monolayer on sulfonated than on nonsulfonated polystyrene. This suggests an unfolded conformation of FN on sulfonated polystyrene, and a more compact one on the nonsulfonated polymer. Significant conformational changes of FN are also indicated by the following: (1) early phase of cell adhesion to FN adsorbed on sulfonated polystyrene surfaces is significantly (ca. factor of 6) higher than to FN on nonsulfonated surfaces, and in the former case adhesion proceeds mostly via alpha(5)beta(1) integrins; (2) RGD, the crucial fragment within central cell binding domain, seems to be partially hidden in the protein structure adopted on nonsulfonated surfaces; (3) patterns of F-actin organization differ in cells adhering to FN on sulfonated and nonsulfonated surfaces. The ELISA study directed against BSA (this protein always present on the surface after the adsorption of FN), showed the importance of "free area," uncovered by both proteins, which influence the cell adhesion processes.     

Surface modification of poly(hydroxybutyrate) films to control cell-matrix adhesion

Tailoring surface properties of degradable polymer scaffolds is key to progress in various tissue engineering strategies. Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thin films were modified by low pressure ammonia plasma, low pressure water vapour plasma, or immersion in a sodium hydroxide solution to elaborate means to control the cell-matrix adhesion of human umbilical cord vein endothelial cells grown on these materials. Fibronectin (FN) heteroexchange and cell adhesion were correlated to the physicochemical characteristics of the modified polymer surfaces which were investigated by X-ray photoelectron spectroscopy (XPS), scanning force microscopy (SFM), electrokinetic measurements, and contact angle measurements. All treatments increased the hydrophilicity of the polymer samples, which could be accounted to newly created amine or carboxyl functionalities for ammonia plasma or water vapour plasma treatments, respectively, and ester hydrolysis for treatments with alkaline aqueous solutions. Main features of cell adhesion and FN reorganisation-evaluated after 1h and after 5 days-could be attributed to the anchorage strength of pre-coated FN layers at the polymer surface, which was, in turn found to be triggered by the type of modification applied. In line with earlier studies referring to different materials cell adhesion and matrix reorganisation were shown to be sensitively controlled through the physicochemical profile of poly(hydroxybutyrate) surfaces.