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Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Sidra Cardiovascular Research

There is increasing evidence for biological functions of human C-peptide. Recently, we have described that proinsulin C-peptide increases nutritive capillary blood flow and restores erythrocyte deformability in type 1 diabetic patients, whereas it has no such effect in non-diabetic subjects. The aim of the current study was to elucidate cellular mechanisms of this vasodilator effect in vitro by measuring the nitric oxide (NO)-mediated increase of cGMP production in a RFL-6 reporter cell assay and by demonstrating endothelial calcium influx with the Fluo-3 technique. C-peptide increased the release of NO from endothelial NO synthase (eNOS) in bovine aortic endothelial cells in a concentration- and time-dependent manner. At physiological concentrations of C-peptide, endothelial NO production was more than doubled (208+/-12% vs control; p<0.001). The NO release was abolished by the inhibitor of NO synthase N(G)-nitro-L-arginine or when Ca(2+) was removed from the medium superfusing the endothelial cells. C-peptide stimulated the influx of Ca(2+) into endothelial cells. No change in Ser-1179 phosphorylation of eNOS was detected after 6.6nM C-peptide. C-peptide did not change eNOS mRNA levels after 1, 6 or 24h. These data indicate that C-peptide is likely to stimulate the activity of the Ca(2+)-sensitive eNOS by increasing the influx of Ca(2+) into endothelial cells. We suggest that this effect may contribute to the increase in skin and muscle blood flow previously demonstrated in human in vivo.

Stimulation of endothelial nitric oxide synthase by proinsulin C-peptide.

Whole human saliva (WHS) and its high molecular weight mucin constituent (Muc) inhibit fibroblast attachment and might influence periodontal and peri-implant wound healing. The aim of this work was to study the potential role of glycosylation of Muc in fibroblast attachment-inhibiting property and to examine in vitro the effect of WHS and Muc on epithelial cell attachment.

Glycosylation pattern and cell attachment-inhibiting property of human salivary mucins.

The aim of this study was to test the applicability of sodium dodecyl sulfate (SDS) agarose gel electrophoresis, electroelution and electrophoretic filtration as methods of separation, detection, and purification of high molecular weight human salivary mucin. SDS agarose gel electrophoresis of whole saliva and mucin prepared by density gradient ultracentrifugation revealed bands with molecular weights in excess of 450 kDa and between 1.6x10(6) and 2x10(6) Da. Electroelution of material from gel and subsequent electrophoresis resulted in highly purified high molecular weight material. Electrophoretic filtration, a method of collecting the material remaining from whole saliva in the slot after penetration of low molecular weight constituents into SDS polyacrylamide gel, failed to produce pure high molecular weight material. It is concluded that SDS agarose gel electrophoresis and electroelution are suitable methods for studying high molecular weight salivary mucin glycoproteins.

Sodium dodecyl sulfate agarose gel electropheresis and electroelution of high molecular weight human salivary mucin.

Modification of material surfaces aimed at bestowing them with antimicrobial properties is a promising approach in the development of new biomaterials. Antimicrobial peptides (AMPs) are an attractive alternative to conventional antibiotics because of lack of toxicity, inherently high selectivity, and absence of immune response. As the antimicrobial mode of action of the AMP cathelin LL37 is formation of pores and disruption of microbial membrane, the purpose of the present study was to develop and test a method of covalent immobilization of LL37 on titanium surface. The application of a flexible hydrophilic poly(ethylene glycol) spacer and selective N-terminal conjugation of LL37 resulted in a surface peptide layer which was capable of killing bacteria on contact.

Preparation of LL-37-grafted titanium surfaces with bactericidal activity.

Direct surface modification of biodegradable polycaprolactone (PCL) was performed without the necessity of synthesis of functionisable co-polymers. An easy-to-perform three-step procedure consisting of amination, reaction with hetero-bifunctional cross-linkers and conjugation of an RGD-motif-containing peptide was used to modify polymer films and improve the attachment of endothelial cells. The biological activity of modified surfaces was assessed by estimating microvascular endothelial cell attachment. Covalent coating with RGD resulted in an approximately 11-fold increase of endothelial cell attachment on modified PCL surfaces compared with untreated polymer. The specificity of the attachment enhancement was confirmed by using a control peptide. It is concluded that chemical surface modification is an appropriate method of rendering degradable polymers, such as PCL, cell-adhesive.

Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films.

Scaffold production for tissue engineering was demonstrated by means of a hot compression molding technique and subsequent particulate leaching. The utilization of spherical salt particles as the pore-forming agent ensured complete interconnectivity of the porous structure. This method obviated the use of potentially toxic organic solvents. To overcome the inherent non-cell-adhesive properties of the hydrophobic polymer polycaprolactone (PCL) surface activation with a diamine was performed, followed by the covalent immobilization of the adhesion-promoting RGD-peptide. The wet-chemical approach was performed to guarantee modification throughout the entire scaffold structure. The treatment was characterized by means of chemical and physical methods with respect to an exclusive surface modification without altering the bulk properties of the polymer. RGD-modified scaffolds were tested in cell-culture experiments to investigate the initial attachment and the proliferation of three different cell types.

Covalent RGD modification of the inner pore surface of polycaprolactone scaffolds.

Polytetrafluoroethylene (PTFE), a frequently utilized polymer for the fabrication of synthetic vascular grafts, was surface-modified by means of a wet-chemical process. The inherently non-cell-adhesive polymer does not support cellular attachment, a prerequisite for the endothelialization of luminal surface grafts in small diameter applications. To impart the material with cell-adhesive properties a treatment with sodium-naphthalene provided a basis for the subsequent immobilization of the adhesion promoting RGD-peptide using a hydroxy- and amine-reactive crosslinker. Successful conjugation was shown with cell culture experiments which demonstrated excellent endothelial cell growth on the modified surfaces.

Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene.

Polyvinyl chloride (PVC) is one of the most frequently used polymers for the manufacturing of medical devices. Limitations for its usage are based upon unfavorable surface properties of the polymer including its hydrophobicity and lack of functionalities in order to increase its versatility. To address this issue, wet chemical modification of PVC was performed through surface amination using the bifunctional compound ethylene diamine. The reaction was conducted in order to achieve maximum surface amination while leaving the bulk material unaffected. The initial activation step was characterized by means of various methods including contact angle measurements, colorimetric amine quantification, infrared spectroscopy, and gel permeation chromatography. Depth profiles were obtained by a confocal microscopic method using fluorescence labeling. Exclusive surface modification was thus confirmed. To demonstrate biological applications of the presented technique, two examples were chosen: The covalent immobilization of the cell adhesive Asp-Gly-Asp-Ser-peptide (RGD) onto PVC samples yielded a surface that strongly supported cellular adhesion and proliferation of fibroblasts. In contrast, the decoration of PVC with the hydrophilic polymer polyethylene glycol prevented cellular adhesion to a large extent. The impact of these modifications was demonstrated by cell culture experiments.

Cell adhesive and antifouling polyvinyl chloride surfaces via wet chemical modification.

In recent years, the synthetic polymer polyetheretherketone (PEEK) has increasingly been used in a number of orthopedic implementations, due to its excellent mechanical properties, bioinertness, and chemical resistance. For in vivo applications, the surface of PEEK, which does not naturally support cell adhesion, has to be modified to improve tissue integration. In the present work we demonstrate a novel wet-chemical modification of PEEK to modify the surface, enabling the covalent grafting of the cell-adhesive RGD-peptide. Modification of the polymer surface was achieved via Schiff base formation using an aliphatic diamine and subsequent crosslinker-mediated immobilization of the peptide. In cell culture experiments with primary osteoblasts it was shown that the RGD-modified PEEK not only significantly promoted cellular adhesion but also strongly enhanced the proliferation of osteoblasts on the modified polymer surface.

Covalent grafting of the RGD-peptide onto polyetheretherketone surfaces via Schiff base formation.

Many biomaterials used for tissue engineering applications lack cell-adhesiveness and, in addition, are prone to nonspecific adsorption of proteins. This is especially important for blood-contacting devices such as vascular grafts and valves where appropriate surface properties should inhibit the initial attachment of platelets and promote endothelial cell colonization. As a consequence, the long-term outcome of the implants would be improved and the need for anticoagulation therapy could be reduced or even abolished. Polytetrafluoroethylene (PTFE), a frequently used polymer for various medical applications, was wet-chemically activated and subsequently modified by grafting the endothelial cell (EC) specific peptide arginine-glutamic acid-aspartic acid-valine (REDV) using a bifunctional polyethylene glycol (PEG)-spacer (known to reduce platelet and nonspecific protein adhesion). Modified and control surfaces were both evaluated in terms of EC adhesion, colonization, and the attachment of platelets. In addition, samples underwent bacterial challenges. The results strongly suggested that PEG-mediated peptide immobilization renders PTFE an excellent substrate for cellular growth while simultaneously endowing the material with antifouling properties.

Matthias Gabriel

Sidra Cardiovascular Research

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion