Surface-immobilized dextran limits cell adhesion and spreading

Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70 kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Immobilized RGD peptides on surface-grafted dextran promote biospecific cell attachment

Dextran has recently been investigated as an alternative to poly(ethylene glycol) (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although antifouling properties of surface-grafted dextran and PEG are quite similar, surface-bound dextran has multiple reactive sites for high-density surface immobilization of biologically active molecules. We recently reported nontoxic aqueous methods to covalently immobilize dextran on material surfaces. These dextran coatings effectively limited cell adhesion and spreading in the presence of serum-borne cell adhesion proteins. In this study we utilized the same nontoxic aqueous methods to graft cell adhesion peptides on low protein-binding dextran monolayer surfaces. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). Surface-grafted cell adhesion peptides stimulated endothelial cell, fibroblast, and smooth muscle cell attachment and spreading in vitro. In contrast, surface-grafted inactive peptide sequences did not promote high levels of cell interaction. Surface-grafted high affinity cyclic RGD peptides promoted cell type-dependent interactions. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

Immobilization of RGD to < 1 1 1 > silicon surfaces for enhanced cell adhesion and proliferation

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and microstructure. One of the greatest challenges with silicon-based biomedical microdevices such as those recently developed for neural stimulation, implantable encapsulation, biosensors, and drug delivery, is to improve biocompatibility and tissue integration. This may be achieved by modifying the exposed silicon surface with bioactive peptides. In this study, Arg-Gly-Asp (RGD) peptide conjugated surfaces were prepared and characterized. The effect of these surfaces on fibroblast adhesion and proliferation was examined over 4 days. Silicon surfaces coupled with a synthetic RGD peptide, as characterized with X-ray photoelectron spectroscopy and atomic force microscopy, display enhanced cell proliferation and bioactivity. Results demonstrate an almost three-fold greater cell attachment! proliferation on RGD immobilized surfaces compared to unmodified (control) silicon surfaces. Modulating the biological response of inorganic materials such as silicon will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.     

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.     

Glial cell and fibroblast cytotoxicity study on plasma-deposited diamond-like carbon coatings

Diamond-like carbon films have been evaluated as coatings to improve biocompatibility of orthopedic and cardiovascular implants. This study initiates a series of investigations that will evaluate diamond-like carbon (DLC) as a coating for improved biocompatibility in chronic neuroprosthetic implants. Studies in this report assess the cytotoxicity and cell adhesion behavior of DLC coatings exposed to glial and fibroblast cell lines in vitro. It can be concluded from these studies that DLC coatings do not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered DLC coatings non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop DLC coatings on prototype microelectrode devices for chronic neural implant applications.     

Titanium with surface-grafted dextran and immobilized bone morphogenetic protein-2 for inhibition of bacterial adhesion and enhancement of osteoblast functions

Failure of bone and joint implants has been attributed mainly to poor bonding of the implant to bone tissue, and to bacterial infection. The probability of successful osseointegration or implant infection depends on the race for the surface between tissue cells and bacteria. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that increases bone cell (osteoblast) function while decreasing bacterial adhesion. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted oxidized dextran, which is known to have activity against bacterial adhesion. Bone morphogenetic protein-2 (BMP-2) was then covalently linked to dextran-grafted surfaces through a chemical conjugation process. The composition and properties of the surface were investigated by X-ray photoelectron spectroscopy and by measuring the surface density of BMP-2 using an enzyme-linked immunosorbent assay. Bacterial adhesion was assayed with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial adhesion on both the dextran and dextran-BMP-2-functionalized surfaces was significantly decreased compared to that on the pristine substrates. Further, the dextran-BMP-2 modified substrates with a surface protein density of >50 ng/cm(2) or higher significantly promoted osteoblast spreading, alkaline phosphatase activity, and calcium mineral deposition. Thus, the results from this study suggest that surface grafting of dextran in conjunction with the bone growth factor BMP-2 on metal surfaces can enhance tissue integration of implants through the dual functions of reducing bacterial adhesion and promoting osteoblast functions.     

Platelet inhibition and endothelial cell adhesion on elastin-like polypeptide surface modified materials

Platelet adhesion and activation are important early markers of biomaterial blood compatibility, while surfaces that promote enhanced endothelial cell adhesion and eNOS expression are strategic targets for long term vascular graft applications. Materials surface modified with fluorinated surface modifiers, containing peptides inspired from elastin cross-linking domains, have been used for the cross-linking of elastin-like polypeptide 4 (ELP4) macromolecules onto polyurethane surfaces. In the present study, ELP4 modified polyurethanes were evaluated in vitro to assess platelet adhesion, microparticle formation and bulk platelet activation following blood-material interactions. Reduced platelet adhesion and bulk platelet activation were observed following contact between reconstituted human blood and the ELP4 materials, relative to the uncoated base polyurethane controls. ELP4 modified materials also promoted endothelial cell adhesion and retention over a period of one week and showed that the endothelial cells exhibited an organized actin cytoskeleton and enhanced endothelial nitric oxide synthase (eNOS) expression relative to the control surfaces. These results indicate that polyurethane elastomers modified with ELP4 covalently bound to fluorinated surface modifiers provide a promising approach for endowing synthetic elastomers with both reduced blood platelet activation properties and enhanced endothelial cell adhesion for potential use in vascular graft applications.     

Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography

Modification of the chemistry and surface topography of nanophase ceramics was used to provide biomaterial formulations designed to direct the adhesion and proliferation of human mesenchymal stem cells (HMSCs). HMSC adhesion was dependent upon both the substrate chemistry and grain size, but not on surface roughness or crystal phase. Specifically, cell adhesion on alumina and hydroxyapatite was significantly reduced on the 50 and 24 nm surfaces, as compared with the 1500 and 200 nm surfaces, but adhesion on titania substrates was independent of grain size. HMSC proliferation was minimal on the 50 and 24 nm substrates of any chemistry tested, and thus significantly lower than the densities observed on either the 1500 or 200 nm surfaces after 3 or more consecutive days of culture. Furthermore, HMSC proliferation was enhanced on the 200 nm substrates, compared with results obtained on the 1500 nm substrates after 7 or more days of culture. HMSC proliferation was independent of both substrate surface roughness and crystal phase. Rat osteoblast and fibroblast adhesion and proliferation exhibited similar trends to that of HMSCs on all substrates tested. These results demonstrated the potential of nanophase ceramic surfaces to modulate functions of HMSCs, which are pertinent to biomedical applications such as implant materials and devices.     

Surface passivation of a microfluidic device to glial cell adhesion: a comparison of hydrophobic and hydrophilic SAM coatings.

Cell adhesion in a microfluidic structure can lead to catastrophic flow problems due to the comparable size of the cell with the microfabricated device. Such issues are important in the growing research area involving the merging of biological materials and MEMS devices. We have examined the surface compatibility of uncoated and coated microfabricated glass and semiconductor surfaces under static solution (cell culture) and flow experiments (microfluidic device) using glial (astrocyte and glioblastoma) cells. Bare semiconductor and glass surfaces were most attractive to cell adhesion, promoting biofouling under both static and flow conditions. Passivation of the surfaces was performed with silane coupling agents octadecyltrimethoxysilane (OTMS) or N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP) on SiO2 surfaces via self-assembled monolayer (SAM) deposition. The hydrophilic TESP coating was effective at inhibiting biofouling of the microfluidic structure, allowing greater than several minutes of fluid flow. The hydrophobic OTMS coating, on the other hand, promoted cell adhesion leading to restricted flow within a few minutes. Interestingly, under cell culture conditions the TESP surface exhibited biocompatible properties for glial cell adhesion and proliferation, in contrast to the OTMS surface which resisted cell growth. These studies suggest that cell adhesion is dependent upon the time domain of the cell-surface interaction.     

Titanium crystal orientation as a tool for the improved and regulated cell attachment

Cell adhesion is a fundamental process that controls cell proliferation, migration, and differentiation and is crucial for biomaterial-tissue integration. Osteoblast attachment on the surfaces of implant materials is, therefore, essential for the proper function of any implant in which osseointegration is required. Although many reports are available on osteoblast attachment using different surface modification, there is no specific report, so far, that investigates the effect of atomic order of specific crystallographic orientation of substrates on cell behavior. A novel coculture system is proposed to show the differential response of preosteoblast and fibroblast cell lines to the titanium single-crystal substrates. Our investigation has shown that surface recognition by the cell is influenced by the atomic structure of the surface leading to cell-type-specific adhesion. The degree of preosteoblast attachment is significantly higher on the Ti-(1120), whereas the fibroblast adhesion is increased on the Ti-(1010). This demonstrates that the three distinct faces of titanium substrates differ greatly in their capacity to serve as cell adhesive substrates. It also provides clear evidence for the role of crystal structure in regulating and improving cell-substrate interactions relevant for the optimal function of bone implant materials.     

Controlled polymerization chemistry to graft architectures that influence cell-material interactions

Acrylate monomers were photografted from polymer substrates to create cell responsive chemically and biologically active surfaces that manipulate cell response. Three monomers, polyethylene glycol monoacrylate (MW 375 g/mol) (PEG375A), a monomeric extra-cellular matrix protein, and a cell-cleavable fluorescent monomer, were spatially photopatterned from a base substrate. The base substrate consisted of a dithiocarbamate (DTC) functionalized urethane diacrylate/tri(ethylene glycol)diacrylate copolymer and was shown to non-specifically support NIH 3T3 fibroblast cell adhesion. The DTC-containing polymer was further modified by grafting PEG375A to demonstrate selective blocking of cell-material interactions. Next, acrylated collagen type I was patterned onto polymer substrates to further promote specific cell interactions (i.e. by presenting cell-adhesive moieties). Hydrophilic PEG375A grafted patterns were shown to prevent 3T3 fibroblast adhesion to polymer in spatially grafted regions, while biologically active acrylated collagen type I promoted cell-surface interactions. Collagen type I was grafted at varying densities (0-7.5 pmol/grafted square), and the extent of cell adhesion and spreading were evaluated for each of these graft densities using fluorescence microscopy. Finally, methacrylated carboxyfluorescein diacetate (CFDA) was synthesized and photografted onto a cell-adhesive substrate as a cell sensing mechanism. The acetate groups found in the structure of CFDA cleave in the presence of cells. This cell-responsive substrate results in fluorescence indicative of acetate-group cleavage associated with cell interactions that occurs in patterned regions on polymer surfaces. Collectively, the results herein show the utility and application of a spatially and temporally controlled photografting process for designing cell responsive polymer surfaces.     

Monocyte/macrophage interactions with base and linear- and star-like PEG-modified PEG-poly(acrylic acid) co-polymers

Poly(ethylene glycol):poly(acrylate) PEG-g-PA co-polymers were made that inhibited nonspecific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides or fragments of antibodies to monocyte/macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Peptides were either directly conjugated to the base material or linked by way of PEO-star tethers. Fragments of the antibody region containing the antigen-binding site (Fab' fragments) were coupled to other PEG-g-PA samples using the sulhydryl end groups on Fab' fragments to amine-bearing PEO stars. Macrophage adhesion rates, phagocytic response (oxidative burst), and cytokine expression were determined for each PEG-g-PA material. Luminol-enhanced chemiluminescence was used as a semiquantitative indication of monocyte-macrophage phagocytic activation (oxidative burst). Macrophage cytokine expression in response to control, base, and modified materials was determined by ELISAs for TNF-alpha, IL-1 beta, IL-6, and IL-8. Tissue culture poly(styrene) (TCPS)-mediated the greatest number of adherent monocyte/macrophage cells relative to PEG-g-PA materials. Both YRGDS and YEILDV peptides, whether directly or indirectly (via StarPEO) conjugated to PEG-g-PA, increased adhesion versus controls. Fab' fragments of all four antibodies also promoted enhanced adhesion versus controls. Fab'StarPEO materials presented two orders of magnitude fewer ligands per surface unit area than peptide star materials (10(8) vs. 10(10)), but were able to adhere similar numbers of cells. For surfaces presenting Fab'(VLA-4) or YEILDV, both of which may both bind to a cell's VLA-4 receptor, the Star:VLA4 surface showed a greater number of adherent monocyte/macrophages. This result suggests that the Fab' had a higher affinity to the cell receptor than a corresponding minimal peptide binding sequence. All materials exhibited low oxidative burst (luminescence counts per minute, LCPM) per cell DNA without the addition of exogenous stimuli (LCPM/DNA < 100). Directly conjugated peptide materials, poly(propylene) (PP), and TCPS showed the lowest levels of LCPM/DNA without the addition of exogenous stimulus (LCPM/DNA < 20). There was no correlation between LCPM/DNA ratios, with and without added LPS stimulus, versus the individual substrates. Monocyte/macrophages adherent to TCPS substrata showed the overall highest stimulatory potential in cytokine expression response to exogenous LPS, followed by PP > PEG-g-PA > StarPEO. Cells adherent to peptide-modified materials and Fab'-modified materials were overall less stimulated. The method of presenting the peptides (i.e., directly or via Star PEO) influenced the level of cytokine secreted by the adherent macrophage.     

Directing cell migration using micropatterned and dynamically adhesive polymer brushes

Micropatterning techniques, such as photolithography and microcontact printing, provide robust tools for controlling the adhesive interactions between cells and their extracellular environment. However, the ability to modify these interactions in real time and examine dynamic cellular responses remains a significant challenge. Here we describe a novel strategy to create dynamically adhesive, micropatterned substrates, which afford precise control of cell adhesion and migration over both space and time. Specific functionalization of micropatterned poly(ethylene glycol methacrylate) (POEGMA) brushes with synthetic peptides, containing the integrin-binding arginine–glycine–aspartic acid (RGD) motif, was achieved using thiol–yne coupling reactions. RGD activation of POEGMA brushes promoted fibroblast adhesion, spreading and migration into previously non-adhesive areas, and migration speed could be tuned by adjusting the surface ligand density. We propose that this technique is a robust strategy for creating dynamically adhesive biomaterial surfaces and a useful assay for studying cell migration.     

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.     

Modulation of cellular responses on engineered polyurethane implants

An in vivo rat cage implant system was used to study the effect of polyurethane surface chemistries on protein adsorption, macrophage adhesion, foreign-body giant cell formation (FBGCs), cellular apoptosis, and cytokine response. Polyurethanes with zwitterionic, anionic, and cationic chemistries were developed. The changes in the surface topography of the materials were determined using atomic force microscopy and the wettability by dynamic contact angle measurements. The in vitro protein adsorption studies revealed higher protein adsorption on cationic surfaces when compared with the base, while adsorption was significantly reduced on zwitterionic (**p < 0.01) and anionic (*p < 0.05) polyurethanes. Analysis of the exudates surrounding the materials revealed no differences between surfaces in the types or levels of cells present. Conversely, the proportion of adherent cells undergoing apoptosis, as determined by annexin V-FITC staining, increased significantly on anionic followed by zwitterionic surfaces (60 + 5.0 and 38 + 3.7%) when compared with the base. Additionally, zwitterionic and anionic substrates provided decreased rates of macrophage adhesion and fusion into FBGCs, whereas cationic surfaces promoted macrophage adhesion and FBGC formation. Visualization of the F-actin cytoskeleton by Alexa Fluor 488 phalloidin showed a significant delay in the cytoskeletal fusion response on zwitterionic and the anionic surfaces. The real-time polymerase chain reaction (PCR) analysis of proinflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-10) and pro-wound healing cytokines (IL-4 and TGF-β) revealed differential cytokine responses. Cationic substrates that triggered stimulation of TNF-α and IL-4 were associated with more spread cells and higher FBGCs, whereas zwitterionic and anionic substrates that suppressed these cytokines levels were associated with less spread cells and few FBGCs. These studies have revealed that zwitterionic and anionic polyurethane surface chemistries can not only reduce nonspecific adhesion, fusion, and inflammatory events but also effectively promote cellular apoptosis in vivo.     

Modulation of the Keratinocyte–Fibroblast Paracrine Relationship with Gelatin-Based Semi-interpenetrating Networks Containing Bioactive Factors for Wound Repair

Gelatin-based semi-interpenetrating networks (sIPNs) containing soluble and covalently-linked bioactive factors have been shown to aid in wound healing; however, the biological responses elicited by the introduction of sIPN biomaterials remain unclear. In the current study, modulation of the re-epithelialization phase of wound healing by sIPNs grafted with PEGylated fibronectin-derived peptides and utilized as platforms for the delivery of exogenous keratinocyte growth factor (KGF) was evaluated. Following wounding, keratinocyte migration, proliferation and protein secretion is largely controlled by diffusible factors, such as KGF, released by the underlying fibroblasts. The impact of sIPNs and exogenous KGF upon the latter keratinocyte–fibroblast paracrine relationship and keratinocyte behavior was explored by monitoring keratinocyte adhesion and cytokine (IL-1α, IL-1β, IL-6, KGF, GM-CSF and TGF-α) release. Results were generally similar for keratinocyte monoculture and keratinocyte–fibroblast co-culture systems. Although keratinocyte adhesion increased over time for positive control surfaces, adhesion to the sIPNs remained low throughout the course of the study. Release of IL-1α and GM-CSF was increased by exogenous KGF. The effects were more noticeable on the positive control surfaces relative to the sIPN surfaces. Regulation of the release of TGF-α was surface dependent, while IL-6 release was dependent upon surface type, the inclusion of exogenous KGF and the presence of fibroblasts. The findings indicate that during re-epithelialization, sIPNs containing soluble bioactive factors aid in wound healing primarily by serving as conduits for KGF, which induces the release of other key cytokines involved in tissue repair.     

Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide

In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates - a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization organization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α(2)β(1) receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α(2)β(1) receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications.     

Novel transparent nano- to micro-heterogeneous substrates for in-situ cell migration study

Transparent substrates having heterogeneities ranging from nanometer to micrometer lateral length scale were fabricated to study cell migration. The surfaces were generated using thin films of block copolymers and homopolymer blends on ultra smooth transparent polyethylene terephthalate films. Results show that the lateral size scale of the surface heterogeneities affects fibroblast (NIH-3T3) adhesion, spreading and motility. More specifically, fibroblasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology on the micron patterned surfaces resemble cells cultured in a 3D environment. These surfaces, therefore, can potentially be utilized as models to study cell behavior in physiologically relevant conditions which can add to our fundamental understanding of cell-substrate interactions and facilitate development of surfaces for medical devices.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.