Analysis of the biological response of endothelial and fibroblast cells cultured on synthetic scaffolds with various hydrophilic/hydrophobic ratios: influence of fibronectin adsorption and conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.     

Effects of plasma treated PET and PTFE on expression of adhesion molecules by human endothelial cells in vitro

The aim of this study was to evaluate the expression of adhesion molecules on the surface of human endothelial cells in response to the systematic variation in materials properties by the ammonia plasma modification of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE). These adhesion molecules act as mediators of cell adhesion, play a role in the modulation of cell adhesion on biomaterials and therefore condition the response of tissues to implants. First and second passage human umbilical vein endothelial cells (HUVECs) were cultured on plasma treated and untreated PET and PTFE. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-alpha) were used as controls. After 1 day and 7 days, the expression of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), Integrin alphavbeta3, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin and L-selectin were evaluated using flow cytometry and immunohistochemistry. There was a slight increase in positive cell numbers expressing the adhesion molecules ICAM-1 and VCAM-1 on plasma treated PET and PTFE. A significant increase in E-selectin positive cells on untreated PTFE was demonstrated after 7 days. Stimulation with TNF-alpha demonstrated a significant increase in the proportion of ICAM-1. VCAM-1 and E-selectin positive cells. Almost all cells expressed PECAM-1 and integrin alphavbeta3, on both materials and controls but did not express P- and L-selectin on any surface. When second passage cells were used, the expression of the adhesion molecules ICAM-1 and VCAM-1 was markedly increased on all surfaces but not with TNF-alpha. These significant differences were not observed in other adhesion molecules. These results were supported by immunohistochemical studies. The effects of plasma treated PET and PTFE on cell adhesion and proliferation was also studied. There was a 1.3-fold increase in cell numbers adhered on ammonia plasma treated PET compared to untreated PET and a 5.5-fold increase in cell numbers on treated PTFE compared to untreated PTFE after 1 day. This is significantly different when analysed statistically. After 7 days, cell number increased significantly on all surfaces compared to 1 day, except for untreated PTFE which conversely reduced by 41%. Cell number on the surface of untreated PET was no different to treated PET on days 1 and 7 when second passage cells were used. The study has shown that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and slightly upregulates the expression of adhesion molecules. This surface modification should promote colonisation of an artificial vascular prosthesis by endothelial cells and make it less vulnerable to immune system cells of the recipient. In addition, it should be considered which passage of cells is used due to the different adhesion features of different passages of HUVECs on untreated PET.     

Development of a YIGSR-peptide-modified polyurethaneurea to enhance endothelialization

Polyurethanes have been investigated for use as vascular grafts due to their excellent mechanical properties and relatively good biocompatibility. However, poor retention of endothelial cells and thrombogenicity in vivo remain problematic for vascular graft applications. The peptide YIGSR has been shown to increase endothelial cell adhesion but not attachment of platelets, suggesting its possible utility for vascular graft applications. In this study, a bioactive polyurethaneurea has been synthesized by incorporating GGGYIGSRGGGK peptide sequences into the polymer backbone. Successful incorporation of the peptides was confirmed by NMR, contact angle measurement and ESCA. Uniform distribution of peptides on the surface was observed using a fluorescent probe capable of reacting with tyrosine residues on the peptides. Hard segment domains were visualized using tapping mode AFM. Endothelial cell adhesion, spreading, proliferation, migration and extra-cellular matrix production were improved on bioactive polyurethaneurea compared to control polyurethaneurea. Competitive inhibition of endothelial cell attachment and spreading by soluble YIGSR peptides indicated that cell adhesion and spreading were specifically mediated by YIGSR-sensitive cell adhesion receptor, not just by changed surface properties. There was no significant difference in the number of adherent platelets. Therefore, this bioactive polyurethanurea may improve vascular graft endothelialization without increasing thrombogenicity.     

Development of a YIGSR-peptide-modified polyurethaneurea to enhance endothelialization

Polyurethanes have been investigated for use as vascular grafts due to their excellent mechanical properties and relatively good biocompatibility. However, poor retention of endothelial cells and thrombogenicity in vivo remain problematic for vascular graft applications. The peptide YIGSR has been shown to increase endothelial cell adhesion but not attachment of platelets, suggesting its possible utility for vascular graft applications. In this study, a bioactive polyurethaneurea has been synthesized by incorporating GGGYIGSRGGGK peptide sequences into the polymer backbone. Successful incorporation of the peptides was confirmed by NMR, contact angle measurement and ESCA. Uniform distribution of peptides on the surface was observed using a fluorescent probe capable of reacting with tyrosine residues on the peptides. Hard segment domains were visualized using tapping mode AFM. Endothelial cell adhesion, spreading, proliferation, migration and extra-cellular matrix production were improved on bioactive polyurethaneurea compared to control polyurethaneurea. Competitive inhibition of endothelial cell attachment and spreading by soluble YIGSR peptides indicated that cell adhesion and spreading were specifically mediated by YIGSR-sensitive cell adhesion receptor, not just by changed surface properties. There was no significant difference in the number of adherent platelets. Therefore, this bioactive polyurethanurea may improve vascular graft endothelialization without increasing thrombogenicity.     

Adhesion and spreading of cultured endothelial cells on modified and unmodified poly (ethylene terephthalate): a morphological study

The in vitro adhesion and spreading of human endothelial cells (HEC) on hydrophobic poly(ethylene terephthalate) (PETP) and moderately wettable tissue culture poly(ethylene terephthalate) (TCPETP) were studied with light microscopy and electron microscopy. Numbers of HEC adhering on TCPETP were always higher than those found on PETP. When cells were seeded in the presence of serum, extensive cell spreading on both PETP and TCPETP was observed after the first 30 min. Thereafter, spread cells appeared to withdraw from the PETP surface, resulting in irregularly shaped cells. Complete cell spreading occurred on TCPETP. Complete cell spreading also occurred on PETP and TCPETP when HEC had first been seeded from phosphate buffer solution and serum was supplied after 30 min. Furthermore, HEC spread on both PETP and TCPETP when the surfaces were precoated with protein(s), which promotes cell adhesion. However, when plasma was used for the coating, spread cells did not proliferate in a monolayer pattern. This study shows that TCPETP is, in general, a better surface for adhesion and proliferation of HEC than is PETP, suggesting that vascular prostheses with a TCPETP-like surface will perform better in vivo than prostheses made of PETP.     

ICAM-1 and VCAM-1 expression by endothelial cells grown on fibronectin-coated TCPS and PS

Small-diameter vascular grafts rapidly fail as a result of blood coagulation and platelet deposition. Endothelial cells lining the inner side of blood vessels can provide the graft lumen with an antithrombogenic surface. One of the remaining problems is cell detachment after restoration of blood flow, because of infiltration of leukocytes that respond to an inflammatory-like activation of the endothelial cells. This endothelial activation is possibly caused by the surface characteristics of the underlying polymer. To get more insight into the effects of the polymer surface on endothelial cell activation, we seeded human umbilical vein endothelial cells (HUVECs) in various densities and subsequently grew them on tissue culture polystyrene (TCPS; hydrophilic) and polystyrene (PS; hydrophobic) surfaces. To improve cell adhesion, surfaces were coated with purified fibronectin prior to cell seeding. During proliferation, the expressions of the leukocyte adhesion molecules ICAM-1 and VCAM-1 were determined. Results indicate that ICAM-1 expression is not influenced by the character of the polymer surface, and that VCAM-1 expression is slightly higher on the TCPS surface. Expressions of both adhesion molecules are influenced by the seeding density and time of proliferation. At low seeding densities (< or = 10,000 cells/cm(2)), a relatively low percentage of nonexogenously activated cells expressed ICAM-1 during the first 3 days of proliferation compared to higher seeding densities. Although less pronounced, this was also observed for the percentage of cells expressing VCAM-1. During proliferation, the amount of ICAM-1 per endothelial cell increased, whereas the expression of VCAM-1 remained low. The absence of large differences in leukocyte adhesion molecule expression by endothelial cells grown on TCPS or PS is possibly caused by coating of the surfaces with fibronectin. It is known that surface hydrophilicity influences protein adsorption. Although this had no or little effect on leukocyte adhesion molecule expression, endothelial cell growth was affected, because proliferation was slower on the hydrophobic PS.     

Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode

The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation.     

Functionalization of poly-(L-lactic-co-epsilon-caprolactone): effects of surface modification on endothelial cell proliferation and hemocompatibility [corrected

A copolymer of L-lactic acid and epsilon-caprolactone (PLLACL) was synthesized with the aim of preparing a bioartificial, small-diameter and partially resorbable vascular graft. The material was submitted to surface functionalizations (i.e. chemical modification by means of hydrolytic 'etching' and plasma discharge) to promote endothelial cell (EC) adhesion and growth avoiding platelet adhesion or coagulation factor absorption. Furthermore, the behaviour of human microvascular endothelial cells (HMVEC) seeded on the untreated and treated copolymer is described, as well as the platelet adhesion and the modifications of coagulation factors determined by the copolymer itself. PLLACL in its native state provided little support for EC adhesion. Improved EC adherence was obtained when functional groups were provided on the polymer surface by surface chemical hydrolysis. HMVEC seeded and cultured on the polymer surface did not show any ultrastructural alteration, thus demonstrating the absence of the polymer cytotoxicity. Moreover, SEM analysis performed on cold plasma modified specimens showed the presence of a subconfluent monolayer of EC, with an elongated spread morphology. Both the untreated and treated copolymers induced only slight variations of platelet number, but determined the activated partial thromboplastin time (APTT) increase, due to factor XI reduction. Finally, a prototype of partially biodegradable vascular prosthesis was prepared with NaOH/HCl-treated copolymer. Pre-cultured HMVEC seeding of the prosthesis by means of a rotation device resulted in an almost completely coverage of the graft inner surface.     

Different growth behaviour of human umbilical vein endothelial cells and an endothelial cell line seeded on various polymer surfaces

Major obstacles for successful application of endothelial cell seeding in synthetic vascular grafts include the source of autologous endothelial cells, the efficiency of cell seeding and detachment of adherent endothelial cells from the graft surface after restoration of circulation. Human umbilical vein endothelial cells (HUVECs) are frequently used to investigate the in vitro adhesion and proliferation of endothelial cells on polymer surfaces. In order to minimize the biological variation of HUVECs isolated from different umbilical veins, it would be advantageous to use an endothelial cell line in in vitro proliferation experiments. Aim of the present study was to compare the proliferation of primary HUVECs and the endothelial cell line EC-RF24 on several polymer surfaces coated with various concentrations of the adhesive protein fibronectin. EC-RF24 cells grow to a higher density than primary HUVECs. Moreover, the EC-RF24 cell line is able to proliferate on surfaces with sub-optimal adhesive properties. Therefore, it is concluded that the EC-RF24 cell line is less suitable for evaluation of the in vitro proliferation of endothelial cells on polymer surfaces.     

Functionalization of poly-L-lactic-co-ε-caprolactone: effects of surface modification on endothelial cell proliferation and hemocompatibility

A copolymer of L-lactic acid and ε-caprolactone (PLLACL) was synthesized with the aim of preparing a bioartificial, small-diameter and partially resorbable vascular graft. The material was submitted to surface functionalizations (i.e. chemical modification by means of hydrolytic 'etching' and plasma discharge) to promote endothelial cell (EC) adhesion and growth avoiding platelet adhesion or coagulation factor absorption. Furthermore, the behaviour of human microvascular endothelial cells (HMVEC) seeded on the untreated and treated copolymer is described, as well as the platelet adhesion and the modifications of coagulation factors determined by the copolymer itself. PLLACL in its native state provided little support for EC adhesion. Improved EC adherence was obtained when functional groups were provided on the polymer surface by surface chemical hydrolysis. HMVEC seeded and cultured on the polymer surface did not show any ultrastructural alteration, thus demonstrating the absence of the polymer cytotoxicity. Moreover, SEM analysis performed on cold plasma modified specimens showed the presence of a subconfluent monolayer of EC, with an elongated spread morphology. Both the untreated and treated copolymers induced only slight variations of platelet number, but determined the activated partial thromboplastin time (APTT) increase, due to factor XI reduction. Finally,a prototype of partially biodegradable vascular prosthesis was prepared with NaOH/HCl-treated copolymer. Pre-cultured HMVEC seeding of the prosthesis by means of a rotation device resulted in an almost completely coverage of the graft inner surface.     

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.     

In vitro study of human vascular endothelial cell function on materials with various surface roughness

In recent years, creating a biodegradable polymer scaffold with an endothelialized surface has become an attractive concept for replacement of small-diameter blood vessels. Toward this end, a better understanding of the interaction between endothelial cells and biodegradable polymer substrates is particularly important. Surface roughness of biomaterials is one of the important parameters that affect cell behavior. In this study, human vascular endothelial cells were cultured on electrospun and solvent-cast poly(L-lactic acid) substrates with different surface roughness. Cell responses were evaluated via both qualitative examinations of cell morphology changes as well as quantitative assessment of cell adhesion and proliferation rate on the different substrates. The results proved that endothelial cell function was enhanced on the smooth solvent-cast surface rather than on the rough electrospun surface of poly(L-lactic acid). Together with our previous findings that electrospun substrates favor vascular smooth muscle cell behavior, it is possible to design a unique three-dimensional scaffold for application of tissue-engineered small-diameter vessel replacement by combining the fabrication technique of solvent casting and electrospinning.     

Effects of base material,plasma proteins and FGF2 on endothelial cell adhesion and growth

Blood-contacting materials rapidly acquire a coating of plasma proteins which can lead to local platelet activation and thrombus formation. This phenomenon seriously limits the usefulness of small diameter synthetic vascular grafts. One solution to this problem is to pre-seed or encourage in situ colonisation of the material with endothelial cells to maintain a non-thrombogenic surface. We have investigated the effect of contact with plasma and serum on the subsequent ability of human endothelial cells to adhere to model hydrophobic and hydrophylic plastic surfaces, and the effect of surface bound fibroblast growth factor 2 (FGF2) on endothelial cell proliferation. Cell adhesion was mainly dependent on adsorbed fibrinogen or vitronectin, depending on the polymer surface, and correlated with antibody binding to these molecules rather than quantitative surface concentrations. Cell proliferation was directly correlated with surface bound FGF2. Surface binding of the latter was controlled both by the chemical nature of the polymer surface and by the presence of FGF-binding molecules adsorbed on the surface. FGF2 bound specifically to surface-adsorbed fibrinogen, fibronectin and vitronectin as well as to pre-coated heparan sulphate proteoglycan, perlecan. Binding was significantly inhibited by plasma and serum which contained high levels of FGF2 binding proteins. To be effective in supporting endothelialisation of vascular grafts in vivo, surface-bound FGF2 would need to be protected from surface dissociation into the circulating blood.     

Hydrodynamic shear-stress-dependent retention of endothelial and endothelial progenitor cells adhered to vascular endothelial growth factor-fixed surfaces

The luminal surfaces of small-diameter artificial vascular grafts must be fully endothelialized to be nonthrombogenic following implantation. To achieve this goal, we have attempted to capture circulating endothelial progenitor cells (EPCs) in situ on the luminal surfaces of implanted grafts. We examined potential receptor-ligand pairs that promote selective and tight adhesion of EPCs using a radial flow chamber comprising three regions, each containing a specific protein-bound substrate: fibronectin (FN) for integrin, and vascular endothelial growth factor (VEGF) and anti-Flk-1 antibody for VEGF receptor. In the presence of shear stress, the greatest retention of endothelial cells and EPCs was observed with FN followed by VEGF and then anti-Flk-1 antibody. Regardless of the bound protein, cell adhesion increased with larger areas of cell adhesion and enhanced cell spreading; the latter was also greatest with FN followed by VEGF and then anti-Flk-1 antibody. The distribution of vinculin-a key protein in focal adhesion plaques-in adherent endothelial cells was examined using total internal reflection fluorescence microscopy; FN-bound surfaces resulted in larger areas of adhesion and more focal adhesion plaques compared with surfaces bound with VEGF. On the other hand, examining these parameters relative to the area of cell adhesion revealed that VEGF-bound surfaces resulted in larger focal adhesion areas and greater fluorescence signals, both of which indicate increased resistance to shear stress. We also discuss in situ capturing of EPCs on surfaces bound with VEGF or anti-Flk-1 antibody, with the goal of creating endothelialized small-diameter vascular grafts.     

Improved endothelial cell adhesion and proliferation on patterned titanium surfaces with rationally designed, micrometer to nanometer features

Previous in vitro studies have demonstrated increased vascular endothelial cell adhesion on random nanostructured titanium (Ti) surfaces compared with conventional (or nanometer smooth) Ti surfaces. These results indicated for the first time the potential nanophase metals have for improving vascular stent efficacy. However, considering the structural properties of the endothelium, which is composed of elongated vascular endothelial cells aligned with the direction of blood flow, it has been speculated that rationally designed, patterned nano-Ti surface features could further enhance endothelial cell functions by promoting a more native cellular morphology. To this end, patterned Ti surfaces consisting of periodic arrays of grooves with spacings ranging from 750 nm to 100 microm have been successfully fabricated in the present study by utilizing a novel plasma-based dry etching technique that enables machining of Ti with unprecedented resolution. In vitro rat aortic endothelial cell adhesion and growth assays performed on these substrates demonstrated enhanced endothelial cell coverage on nanometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, as well as controls consisting of random nanostructured surface features. Furthermore, nanometer-patterned Ti surfaces induced endothelial cell alignment similar to the natural endothelium. Since the re-establishment of the endothelium on vascular stent surfaces is critical for stent success, the present study suggests that nanometer to submicrometer patterned Ti surface features should be further investigated for improving vascular stent efficacy.     

Improving arterial prosthesis neo-endothelialization: application of a proactive VEGF construct onto PTFE surfaces

The formation of a confluent endothelium on expanded polytetrafluoroethylene (PTFE) vascular prostheses has never been observed. This lack of endothelialization is known to be one of the main reasons leading to the development of thromboses and/or intimal hyperplasia. In this context, several efforts were put forward to promote endothelial cell coverage on the internal surface of synthetic vascular prostheses. The goal of the present study was to immobilize the vascular endothelial growth factor (VEGF) onto Teflon PTFE surfaces to generate a proactive polymer construct favoring interaction with endothelial cells. An ammonia plasma treatment was first used to graft amino groups on PTFE films. Subsequent reactions were performed to covalently bind human serum albumin (HSA) on the polymer surface and to load this protein with negative charges, which allows adsorbtion of VEGF onto HSA via strong electrostatic interactions. X-ray photoelectron spectroscopy (XPS) experiments along with surface derivatization strategies were performed between each synthesis step to ascertain the occurrence of the various molecules surface immobilization. Finally, the electrostatic binding of VEGF to the negatively charged HSA matrix was performed and validated by ELISA. Endothelial cell adhesion and migration experiments were carried out to validate the potential of this VEGF-containing biological construct to act as a proactive media toward the development of endothelial cells.     

High-affinity augmentation of endothelial cell attachment: long-term effects on focal contact and actin filament formation

Coadsorption of high-affinity avidin with lower affinity cell adhesion protein fibronectin has been shown to significantly augment short-term (1 h) adhesion and spreading of endothelial cells; however, the longer term persistence of avidin binding and its effect on endothelial cell adhesion have not been addressed. In this study, the presence of avidin-biotin bonds 24 h after cell adhesion to the dual ligand surfaces was verified by laser confocal microscopy of a fluorescent avidin analog, streptavidin. Total internal reflection microscopy showed that the focal contact area, focal contact density, and cell spreading all increased significantly at 24 h compared to fibronectin-treated control surfaces. Focal contact area was identical when measured with cells that were labeled with either the fluorescent streptavidin or a carbocyanine dye incorporated in the cell membrane. Confocal images of stress fibers formed in cells adherent to dual ligand surfaces after 24 h were thicker and more numerous compared to cells adherent to fibronectin controls. The results indicate that 24 h after initial attachment avidin-biotin is localized to focal contacts on the basal surface and affects cell spreading, actin filament organization, and focal contact density.     

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.     

Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers

The interaction of cells with solid surfaces is important in many settings, including the response of tissue to implanted materials. Protein adsorption to the surface plays a critical role in controlling cell interactions with surfaces. However, few comprehensive studies of both cell behavior and protein adsorption in complex protein mixtures (e.g., serum) have been done so the connection between these events is not well understood. In particular, methods to systematically perturb both protein adsorption and cell behavior in order to understand their relationship have been lacking. To induce changes in cell and protein behavior, the effects of serum dilution and substrate surface chemistry were studied. Surface chemistry was varied by using a series of polymers and copolymers of hydroxyethyl methacrylate (HEMA) and ethylmethacrylate (EMA) varying in their hydrophobic/hydrophilic balance. Large changes in cell spreading and fibronectin adsorption were observed when either serum concentration or polymer type was varied. The spreading of 3T3 cells in serum was found to be well correlated with the amount of fibronectin adsorption to the substrates. Attachment was not correlated with fibronectin adsorption, especially on glass preadsorbed with diluted serum. For 3T3 cells and perhaps other cells that have a receptor for a protein which is present in the medium, the amount of adsorption of this protein to the substrate appears to be a critical factor controlling cell interactions with the substrate.     

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.