In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.     

Stable immobilization of rat hepatocytes as hemispheroids onto collagen-conjugated poly-dimethylsiloxane (PDMS) surfaces: importance of direct oxygenation through PDMS for both formation and function

The highly oxygen-permeable material, poly-dimethylsiloxane (PDMS), has the potential to be applied to cell culture microdevices, but cell detachment from PDMS has been a major problem. In this study, we demonstrate that a combination of collagen covalently immobilized PDMS and an adequate oxygen supply enables the establishment of a stable, attached spheroid (hemispheroid) culture of rat hepatocytes. The bottom PDMS surfaces were first treated with oxygen plasma, then coupled with aminosilane followed by a photoreactive crosslinker, and they were finally reacted with a collagen solution. X-ray photoelectron spectroscopy (XPS) and contact angle measurements showed that the covalent immobilization of collagen on the surface occurred only where the crosslinker had been introduced. On the collagen-conjugated PDMS surface, rat hepatocytes organized themselves into hemispheroids and maintained the viability and a remarkably high albumin production at least for 2 weeks of culture. In contrast, hepatocytes on the other types of PDMS surfaces formed suspended spheroids that had low albumin production. In addition, we showed that blocking the oxygen supply through the bottom PDMS surface inhibited the formation of hemispheroids and the augmentation of hepatocellular function. These results show that appropriate surface modification of PDMS is a promising approach towards the development of liver tissue microdevices.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

A蛋白定向固定抗体用于椭偏光学生物传感器免疫检测

椭偏光学生物传感器是在椭偏光学显微成像技术的基础上发展的一项生物传感技术。它能够直接观测固体表面上的生物分子面密度,毋需任何标记辅助,适合发展成为一种无标记免疫检测技术。研究了在硅片表面上通过A蛋白定向固定抗体分子用于椭偏光学生物传感器免疫检测的可能性。实验结果表明,通过A蛋白固定抗体得到的抗体膜层的均一性和固定量的重复性能够保证椭偏光学生物传感器免疫检测结果的质量。通过A蛋白定向固定的抗体的抗原结合位点趋向一致,显著提高了抗体与抗原结合的能力。此外,通过蛋白A固定的免疫球蛋白G分子能够结合更多的多克隆抗体分子说明通过A蛋白固定的蛋白质分子能够较好地保持其空间构象。     

Osteogenic Surface Modification Based on Functionalized Poly-P-Xylylene Coating

The biotechnology to immobilize biomolecules on material surfaces has been developed vigorously due to its high potentials in medical applications. In this study, a simple and effective method was designed to immobilize biomolecules via amine-N-hydroxysuccinimide (NHS) ester conjugation reaction using functionalized poly-p-xylylene coating on material surfaces. The NHS ester functionalized coating is synthesized via chemical vapor deposition, a facile and solvent-less method, creating a surface which is ready to perform a one-step conjugation reaction. Bone morphogenetic protein 2 (BMP-2) is immobilized onto material surfaces by this coating method, forming an osteogenic environment. The immobilization process is controlled at a low temperature which does not damage proteins. This modified surface induces differentiation of preosteoblast into osteoblast, manifested by alkaline phosphatase (ALP) activity assay, Alizarin Red S (ARS) staining and the expression of osteogenic gene markers, Alpl and Bglap3. With this coating technology, immobilization of growth factors onto material surface can be achieved more simply and more effectively.     

Optimization of ultraviolet ozone treatment process for improvement of polycaprolactone (PCL) microcarrier performance

Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the surface functionality of certain polymeric microcarriers for effective cell attachment and growth remains a challenge. Polycaprolactone (PCL), a biodegradable polymer has received considerable attention due to its good mechanical properties and degradation rate. The drawback is the non-polar hydrocarbon moiety which makes it not readily suitable for cell attachment. This report concerns the modification of PCL microcarrier surface (introduction of functional oxygen groups) using ultraviolet irradiation and ozone (UV/O₃) system and investigation of the effects of ozone concentration, the amount of PCL and exposure time; where the optimum conditions were found to be at 60,110.52 ppm, 5.5 g PCL and 60 min, respectively. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.92 nmol/g and the amount of gelatin immobilized was 320 ± 0.9 µg/g on UV/O₃ treated microcarriers as compared to the untreated (26.83 ± 3 µg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with the attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93°–49.34°) after UV/O₃ treatment and subsequently after immobilization of gelatin. The attachment and growth kinetics for HaCaT skin keratinocyte cells showed that adhesion occurred much more rapidly for oxidized surfaces and gelatin immobilized surface as compared to untreated PCL.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan-gelatin porous structures

Influence of mechanical characteristics and matrix architecture of substrates used in cell culture is an important issue to tissue engineering. Chitosan‐based materials have been processed into porous structures, injectable gels and membranes, and are investigated to regenerate various tissues. However, the effect of these structures on cell growth and matrix production in accordance with each of the differing scaffolds has not been examined. We investigated the influence of porous structures, hydrogels, and membranes on the growth of normal human fibroblasts and their matrix production in a serum‐free system. We used chitosan alone and in combination with gelatin. Injectable hydrogels were prepared using 2‐glycerol phosphate. From the same solution, porous scaffolds and membranes were formed using controlled rate freezing and lyophilization, and air‐drying, respectively. Fibroblast growth was evaluated on the 4th and 10th days using flow cytometry and CFDA‐SE pre‐staining. Cell morphology was assessed using actin and nucleus staining. Total protein content, collagen, tropoelastin, and MMP2/MMP‐9 activity in the media supernatant were assessed by BCA, Sircol?, Fastin Elastin, and fluorogeneic peptide assays. Collagen accumulated in the matrix was assessed by Sircol? assay after pepsin/acetic acid digestion and by Masson's Trichrome staining. These results showed increased viability of fibroblasts on chitosan–gelatin porous scaffold with decreased proliferation relative to tissue culture plastic (TCP) surface despite the cells showing spindle shape. The total protein, collagen, and tropoelastin contents were higher in the spent media from chitosan–gelatin porous scaffolds compared to other conditions. MMP2/MMP9 activity was comparable to TCP. An increase in collagen content was also observed in the matrix, suggesting increased matrix deposition. In summary, matrix production is influenced by the form of chitosan structures, which significantly affects the regenerative process. Biotechnol. Bioeng. 2012; 109:1314–1325. © 2011 Wiley Periodicals, Inc.     

Cultured skin cells interaction with polylactide surface coated by different collagen structures

The purpose of this work was an optimization of polylactide film surfaces designed for human keratinocytes cultivation. The polylactide films were coated by collagen 1. The experiments showed that uniform covering of polymer surface by collagen, and formation of different collagen structures depend on the mode of the protein application. The differences in collagen distribution on the polymer surface influened the keratinocytes growth in culture. Analysis of keratinocytes alignment, as well as cytoskeleton organization demonstrated that fibrillar collagen promoted more even keratinocytes distribution in comparison with the distribution on molecular collagen.     

Cell-surface charge and cell-surface hydrophobicity of collagen-bindingAeromonas andVibrio strains

Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A. caviae, A. sobria, Vibrio cholerae, and V. anguillarum strains. These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the α1+α2 chains) and gelatin immobilized on latex beads. Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties. There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen. The expression of surface properties was dependent on the culture media. There was no relationship between binding to immobilized collagen and binding to soluble ¹²⁵I-labeled collagen. Particle-agglutination reactivity differed when using various collagen-coated microbead preparations. There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures. The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran. Under these conditions, the α1+α2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads. For latex beads passively coated with collagen preparations, the α1+α2 collagen-chain mixture was more hydrophobic than gelatin and native collagen. We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads. In this regard, carboxy-modified latex beads coated with an α1+α2 collagen-chain mixture gave the best results. Received: 9 January 1995 / Accepted: 30 May 1995     

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner aggrecan was studied by comparing surface plasmon resonance to native HA; this shows that binding of aggrecan was achieved in a similar way. Dermal human fibroblasts were used as a model cell to study how chemical modification and immobilization of HA impact adhesion and spreading of cells, which also affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification.     

Photo-immobilization of biological components on gold-coated chips for measurements using surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM)

The photo-immobilization technique is useful for immobilization of various biomolecules on assorted material surfaces, independent of the organic functional groups that may be present. Here, we report a convenient new photo-immobilization technique that was developed by combining a nonbiofouling polymer containing polyethylene glycol and a photoreactive crosslinker for surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. By this method, nonspecific interactions were reduced and various types of molecules, bovine serum albumin, heparin, dsDNA, phosphatidylserine, Tobacco Mosaic Virus, and norfloxacine, were immobilized on an alkane thiol-modified gold surface by a single method. The interactions of photo-immobilized biomolecules and their corresponding antibodies were investigated by SPR and QCM. In addition, SPR imaging was possible using the present method.     

Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate.

A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules.     

Patterning of cells on functionalized poly(dimethylsiloxane) surface prepared by hydrophobin and collagen modification

Controllable cell growth on poly(dimethylsiloxzne) (PDMS) surface is important for its potential applications in biodevices. Herein, we developed a fully biocompatible approach for patterning of cells on the PDMS surface by hydrophobin (HFBI) and collagen modification. HFBI and collagen were immobilized on the PDMS surface one after another by using copper grids as a mask. HFBI self-assembly on PDMS surface converted the PDMS surface from hydrophobic to hydrophilic, which facilitated the following immobilization of collagen. Collagen had admirable ability to support cell adhesion and growth. Consequently, the HFBI/collagen-modified PDMS surface could promote cell adhesion and growth. What is more, the native PDMS surface did not support cell adhesion and growth. Patterning of cells was achieved by directly culturing 293T cells (the human embryonic kidney cell line) on the PDMS surface patterned with HFBI/collagen. Further studies by means of gene transfection experiment in vitro showed that the patterned cells were of good bioactivities. Herein, the biocompatible preparation of cell patterns on the PDMS surface could be of many applications in biosensor device fabrication.     

Novel potentiometric immunosensor for hepatitis B surface antigen using a gold nanoparticle-based biomolecular immobilization method

A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles, Nafion, and gelatin as matrices in this study. The modification procedure of the immunosensor was further characterized by using cyclic voltammetry and the enzyme-linked immunosorbent assay (ELISA) method. The detection is based on the change in the electric potential before and after the antigen-antibody reaction. In contrast to the commonly applied methods (e.g., the glutaraldehyde crosslinking procedure), this strategy could allow for antibodies immobilized with a higher loading amount and better retained immunoactivity, as demonstrated by the potentiometric measurements. A dynamic concentration range of 4-800 ng ml(-1) and a detection limit of 1.3 ng ml(-1) were observed. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by ELISA. In addition, the technique presents some distinct advantages over the traditional sandwich format in that the analyzing performances are direct, rapid, and simple without multiple separation and labeling steps.     

Collagen adsorption on quercetin loaded polycaprolactone microspheres: approach for "stealth" implant

We recently experimented with collagen coating on the surface of quercetin loaded polycaprolactone microspheres by simple adsorption technique to mimic extra cellular matrix and reduce immune or inflammatory responses at the site of implants. The collagen immobilization on polymeric scaffold surfaces through various surface modification techniques was the current scenario to improve bio-integration of the polymers with the in vivo system. Nevertheless, it requires other chemicals or processing methods to modify the surface of polymers to immobilize the collagen covalently. Here protein adsorption principle is used for the coating of collagen onto the surface of solid microspheres and characterized. Optical, ATR-FTIR, SEM analysis confirm collagen coating. The reduction in burst release of the quercetin from the PCL microspheres further confirms its presence and role in the controlled release. The results indicate that the adsorption technique can be the simple strategy to coat collagen on the surface of polyester implants to develop stealth implant in shorter time with low cost technology.     

A strategy for the covalent functionalization of resorbable polymers with heparin and osteoinductive growth factor

The chemical strategy presented herein is the nondestructive preparation of resorbable polymer scaffolds with heparin covalently bonded to the surface and an osteoinductive growth factor, recombinant human bone morphogenetic protein-2, immobilized in the heparin layer. The coupling scheme involves functionalization of surfaces by grafting in the vapor phase with poly( l-lactide) and poly(-caprolactone) films chosen as representative substrates. The biocompatibility of functionalized surfaces was verified by a much improved attachment and proliferation of mesenchymal stem cells (MSC).     

PCL film surfaces conjugated with P(DMAEMA)/gelatin complexes for improving cell immobilization and gene transfection

Successful gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration by enabling the localized production of therapeutic drugs. Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but its unfavorable cell-adhesion property needs to be improved. In this work, the PCL film surface was conjugated with poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA))/gelatin complexes via surface-initiated atom transfer radical polymerization (ATRP) for improving cell immobilization and subsequent gene transfection. A simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL film. Well-defined P(DMAEMA) brushes were subsequently prepared via surface-initiated ATRP from the initiator-functionalized PCL surfaces. The P(DMAEMA) chains with a pK(a) of 7.0-7.3 were used for conjugating gelatin with a pI of 4.7 via electrostatic interaction. The amount of complexed gelatin increased as that of the grafted P(DMAEMA) layer. The cell-adhesion property on the functionalized PCL surface could be controlled by adjusting the ratio of P(DMAEMA)/gelatin. It was found that the gene transfection property on the immobilized cells was dependent on the density of the immobilized cells on the functionalized PCL film. With the good cell-adhesive nature of gelatin and the efficient gene transfection on the dense immobilized cells, the incorporating the suitable of P(DMAEMA)/gelatin complexes onto PCL surfaces could endow the PCL substrates new and interesting properties for potential tissue engineering applications.     

Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Modification of a traditional live–dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface‐immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3‐(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si‐QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si‐QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si‐QAC surfaces showed no kill after removing all Si‐QAC elutables via rinsing procedures. The semi‐quantitative surface‐separated live–dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents. Biotechnol. Bioeng. 2011; 108:231–236. © 2010 Wiley Periodicals, Inc.