Surface Modification of Polytetrafluoroethylene and Polycaprolactone Promoting Cell-Selective Adhesion and Growth of Valvular Interstitial Cells.

Tissue engineering concepts, which are concerned with the attachment and growth of specific cell types, frequently employ immobilized ligands that interact preferentially with cell types of interest. Creating multicellular grafts such as heart valves calls for scaffolds with spatial control over the different cells involved. Cardiac heart valves are mainly constituted out of two cell types, endothelial cells and valvular interstitial cells. To have control over where which cell type can be attracted would enable targeted cell settlement and growth contributing to the first step of an engineered construct. For endothelial cells, constituting the outer lining of the valve tissue, several specific peptide ligands have been described. Valvular interstitial cells, representing the bulk of the leaflet, have not been investigated in this regard. Two receptors, the integrin α9ß1 and CD44, are known to be highly expressed on valvular interstitial cells. Here, we demonstrate that by covalently grafting the corresponding peptide and polysaccharide ligand onto an erodible, polycaprolactone (PCL), and a non-degradable, polytetrafluoroethylene (PTFE), polymer, surfaces were generated that strongly support valvular interstitial cell colonization with minimal endothelial cell and reduced platelet adhesion. The technology for covalent binding of corresponding ligands is a key element towards tissue engineered cardiac valves for in vitro applications, but also towards future in vivo application, especially in combination with degradable scaffold material.

Inactivation of an Enveloped Virus by Immobilized Antimicrobial Peptides.

Infections caused by viruses are difficult to treat due to their life cycle, which depends on the replication machinery of the respective host cells. Commonly used antiviral strategies are based upon the application of, e.g., entry inhibitors and other compounds that interfere with virus replication. Besides possible side effects, the rapid occurrence of viral resistance poses a great challenge. Antimicrobial peptides (AMPs), as a component of the innate immunity, are able to kill bacteria and fungi and, in addition, may inactivate enveloped viruses. Many AMPs exert their biological function by impairing microbial and viral membranes. As a result, membrane integrity is lost, leading to bacterial killing and virus inactivation. Covalently immobilized AMPs have been shown to be biocidal too, which is of special interest when the presence of a soluble agent is to be avoided. Here, we demonstrate the conjugation of the human AMP LL37 to a solid support consisting of cellulose beads and its capability to inactivate murine cytomegalovirus as an example. Virus inactivation was highly reduced by several orders of magnitude when an appropriate coupling strategy was chosen. Coupling the AMP via a long and hydrophilic polyethylene glycol spacer proved to perform less effective compared to LL37 immobilization using a short cross-linker. In addition, it was found that LL37-conjugated beads did not induce hemolysis, a prerequisite for the development of blood contacting applications. Our findings may serve as a basis for the development of an implementable device that is able to reduce the viral load under real-life conditions.

Combined Endothelialization Promoting and Surface Binding Chimeric Conjugate with Low Thrombogenicity.

Endothelialization of blood contacting implants, e.g., vascular stents, is regarded as a prerequisite for an improved performance in terms of minimizing thrombogenicity and the inhibition of restenosis. Commonly used materials, such as Ti-based alloys, can be surface-modified in order to improve endothelial cell (EC) colonization as well as to reduce platelet adhesion. Standard modification techniques involve silanization and are laborious and time-consuming. We propose a novel single-step procedure based on a surface-recognizing peptide generated by phage display methodology. Combining this with a polyethylene glycol (PEG) spacer and an EC-specific sequence yielded a conjugate applicable for the modification of Ti surfaces.

The impact of different low-pressure plasma types on the physical, chemical and biological surface properties of PEEK.

OBJECTIVE: Plasma treatment can be used as surface treatment of PEEK (poly-ether-ether-ketone) to increase the bonding strength between veneering composite and dental prosthetic frameworks of PEEK or enhance biocompatibility of PEEK implants. These improvements are probably based on chemical changes of the PEEK surface. However, the aim of the study was to evaluate the impact of different low-pressure plasma treatments on surface properties of PEEK, such as roughness, hydrophilicity, micro-hardness, crystallinity and biological activity of PEEK. METHODS: Due to different plasma treatments, 143 disc-shaped specimens of pure implantable PEEK were divided into 4 groups: PEEK (no plasma treatment, n = 29), H-PEEK (hydrogen plasma treatment, n = 38), O-PEEK (oxygen plasma treatment, n = 38), H/O-PEEK (hydrogen/oxygen plasma treatment with a gas mix ratio of 2:1, n = 38). Subsequently, surface roughness, surface contact angle, surface crystallinity, surface micro-hardness and human osteoblast cell coverage area of each group were examined. RESULTS: The hydrophilicity, crystallinity and micro-hardness of the plasma-treated groups increased significantly compared to the untreated group, whereas significant differences in the results of the micro-hardness tests could be shown between all groups up to a test force of 0.02N. Cell density was significantly higher on treated vs. untreated PEEK surfaces. Oxygen and H/O plasma treatments revealed to be most effective, whereas H/O plasma worked ten times faster to achieve the same effects. SIGNIFICANCE: The hydrogen-oxygen, 2/1-mixed plasma treatment combines the effect of hydrogen and oxygen plasma which strongly improve the surface properties of PEEK implant material, such as hydrophilicity, crystallinity, surface micro-hardness and HOB cell adhesion.

Manipulation of cellular behaviour on surface-modified polyvinylidene difluoride using wet chemistry.

The surface modification of polyvinylidene difluoride (PVDF) for various biomedical uses is notoriously hampered by the chemical inertness of the polymer. A wet chemical approach aiming at covalently grafting biomolecules was demonstrated by means of an elimination reaction of fluorine from the polymer backbone followed by subsequent modification steps. Exemplified as a possible biological application, the coupling of the peptide REDV rendered the material adhesive for endothelial cells while adhesion of thrombocytes was dramatically reduced.

The covalently immobilized antimicrobial peptide LL37 acts as a VEGF mimic and stimulates endothelial cell proliferation.

The chemical coupling of growth factors to solid substrates are discussed as an alternative to delivery systems. Utilizing entire proteins for this application is hampered by safety and stability considerations. Instead, growth factor mimicking peptides are of great interest for biomedical applications, such as tissue engineering, due to their purity and stability. The human cathelicidin derived antimicrobial peptide LL37, beside its microbicidal activity, was shown to stimulate endothelial cell growth when used in a soluble form. Here, in a novel approach, spacer mediated immobilization, but not direct conjugation of LL37, to a gold substrate was shown to result in a pronounced mitogenic effect on endothelial cells, comparable to that of soluble vascular endothelial growth factor.

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 interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface. This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Tailoring Novel PTFE Surface Properties: Promoting Cell Adhesion and Antifouling Properties via a Wet Chemical Approach.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

BACKGROUND: 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. METHODS: Muc was isolated from WHS by CsCl density gradient ultracentrifugation; covalently immobilized on polystyrene; and subjected to enzymic digestion by N-glycanase, O-glycanase, and sialidase, or chemical desulfation and periodate treatment. Wells of tissue culture microtiter plates were incubated with WHS, Muc, or buffer as control; suspensions of normal human oral keratinocytes, spontaneously immortalized human keratinocytes, or human gingival fibroblasts were applied; and cell attachment determined using methylene blue assay. RESULTS: While enzymic cleavage of N-linked carbohydrates showed no effect, selective removal of O-linked residues and sialic acid as well as desulfation and periodate oxidation resulted in statistically significant reduction of cell attachment-inhibiting property of immobilized Muc. Significantly lower numbers of attached cells of each cell type were found in wells pretreated with WHS or Muc. CONCLUSIONS: Inhibition of cell attachment may be mediated by the carbohydrate residues suggesting specific interactions between the salivary constituent and the cell surface. Exposure of root and implant surfaces to saliva during early wound healing events might influence healing by inhibiting surface colonization by oral keratinocytes and fibroblasts, and enhancing wound repair in the form of long junctional epithelium rather than regeneration.

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

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.