Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine–glycine–aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell–biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.     

Dhvar5 antimicrobial peptide (AMP) chemoselective covalent immobilization results on higher antiadherence effect than simple physical adsorption

Bacterial colonization and subsequent biofilm formation is still one of the major problems associated with medical devices. Antimicrobial peptides (AMP) immobilization onto biomaterials surface is a promising strategy to avoid bacterial colonization. However, a correct peptide orientation and exposure from the surface is essential to maintain AMP antimicrobial activity.This work aims to evaluate the effect of the immobilization on antibacterial activity of Dhvar5 (LLLFLLKKRKKRKY), an AMP with a head-to-tail amphipathicity. Dhvar5 was linked to thin chitosan coatings in i) a controlled orientation and exposure, testing covalent immobilization of its N- or C-terminus and using spacers with different lengths and flexibilities or in ii) a random orientation by physical adsorption. Chitosan coating was chosen due to its antimicrobial properties and readiness to be functionalized.Surface characterization demonstrated the chemoselective immobilization of the peptide with different spacers in a similar concentration (∼2 ng/cm²).Efficacy assays demonstrated that covalent immobilization of Dhvar5 exposing its cationic end, improves the chitosan coating antimicrobial effect by decreasing Methicillin-resistant Staphylococcus aureus (MRSA) colonization. This effect was enhanced when longer spacers were used independently of their flexibility. In opposite, immobilized Dhvar5 exposing its hydrophobic end has no effect on bacterial adhesion to chitosan, and when adsorbed in a random orientation even induces bacterial adhesion to chitosan coating.     

Surface functionalization of hyaluronic acid hydrogels by polyelectrolyte multilayer films

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.     

Biomedical coatings based on chitin soluble extract for inhibition of fungal adhesion to polymeric surfaces

Indwelling medical devices made of polymeric materials, such as intravenous (IV) catheters, are known risk factors for development of fungal infection, particularly systemic candidiasis, that are a significant cause of morbidity and lethality in compromised patients. Candida can form a biofilm on the polymeric surface, serving as a nidus for systemic, difficult to eradicate infection. The current research focuses on development and study of a chitin soluble extract (CSE) coating on polyurethane (PU), in order to reduce the level of adherence of C. albicans to the PU surface. The immobilization of CSE onto the PU surface was performed using both, chemical binding and physical adsorption. Our results indicate that CSE develops a unique tertiary structure in which basic elements in the range of 100 nm build "fingers" and these "fingers" are arranged in a concentric structure around a center. The CSE coated films showed 75% inhibition of C. albicans adhesion for the chemical binding and 83% for the physical adsorption coating and even after 11 weeks the inhibitory effect on adhesion is still significant. Hence, our new coatings may lead to a new generation of medical devices with surfaces that can prevent fungal adhesion.     

Novel synthetic anti-fungal tripeptide effective against Candida krusei

Introduction: Candida species are the major fungal pathogens of humans. Among them, Candida krusei have emerged as a notable pathogen with a spectrum of clinical manifestations and is known to develop resistance against azoles mainly fluconazole. Anti-microbial peptides play important roles in the early mucosal defence against infection and are potent anti-fungal agents since they fight against fungal infection as well as have ability to regulate host immune defence system. The aim of the study was to synthesize a small anti fungal peptide. Materials and Methods: The series of tripeptides were synthesized and screened for antifungal activity against Candida strains according to CLSI guidelines. Toxicity effect of peptide was tested with human erythrocytes. The mode of action of peptide on fungus was resolved by scanning electron microscopy (SEM) studies Results: The tripeptide FAR showed a prominent anti fungal activity among the series. The minimum inhibitory concentration and minimum fungicidal concentration of tripeptide FAR was found to be 171.25 μg/ml and 685 μg/ml, respectively against Candida krusei. The therapeutic index was 2.9. The haemolytic experiment revealed that this peptide is non - toxic to human cells. The SEM studies showed disruption of cell wall and bleb-like surface changes and irregular cell surface. Conclusion: The peptide showed a significant antifungal activity against C. krusei. Thus, it can set a platform for the design of new effective therapeutic agents against C. krusei.     

Echinocandins: Their Role in the Management of Candida Biofilms

The importance of antifungal agents and their clinical implications has received little attention in comparison to antibiotics, particularly in the health-care setting. However, apart from bacterial infections rising in hospitals, the incidences of fungal infections are growing with the development of resistance to conventional antifungal agents. Newer antifungal agents such as echinocandins (ECs) have been extensively studied over the past decade and are recognised as a superior treatment compared with prior antifungals as a first line of therapy in tertiary institutions. Caspofungin (CAS), micafungin (MICA) and anidulafungin (ANID) are the three most widely used EC antifungal agents. The treatment of biofilm-associated fungal infections affecting patients in tertiary health-care facilities has been identified as a challenge, particularly in Indian Intensive Care Unit (ICU) settings. With the rising number of critically ill patients requiring invasive devices such as central venous catheters for treatment, especially in ICUs, these devices serve as a potential source of nosocomial infections. Candida spp. colonisation is a major precursor of these infections and further complicates and prolongs treatment procedures, adding to increasing costs both for hospitals and the patient. Analysing studies involving the use of these agents can help in making critical decisions for antifungal therapy in the event of a fungal infection in the ICU. In addition, the development of resistance to antifungal agents is a crucial factor for assessing the appropriate antifungals that can be used for treatment. This review provides an overview of ANID in biofilms, along with CAS and MICA, in terms of clinical efficacy, resistance development and potency, primarily against Candida spp.     

Relevance of bi-functionalized polyelectrolyte multilayers for cell transfection

In an effort to develop new biomaterial coatings, it was shown that polyelectrolyte multilayers constitute a very powerful tool to render surfaces biologically active. The challenge is to multi-functionalize surfaces in a controlled way. We show here, for the first time, that it is possible to functionalize multilayer films simultaneously with two molecules acting in totally different ways on cells, namely plasmid DNA (pDNA), pre-complexed with poly(ethyleneimine) (PEI), and a peptide molecule, NDPMSH. This peptide, grafted to poly(L-glutamic acid) (PGA) was used as a signal molecule for melanoma cells B16-F1 and for its ability to enhance gene delivery in a receptor-independent manner. The PGA-NDPMSH chains are embedded in poly-(allylamine hydrochloride)/poly-(sodium 4-styrene sulfonate) multilayers and the pDNA-PEI complexes are deposited on top of the films. It is shown that melanoma cells (B16-F1) are efficiently transfected after 24h of contact with functionalized films. When brought in contact with Huh-7 cells that do not express the peptide receptors, these films trigger significantly the transfection rate, showing that it is possible to enhance the transfection process by incorporating specific peptides into multilayer films. Moreover, transfected cells sorted by flow cytometry produce melanin, demonstrating both activation via the peptide signaling pathway and cell transfection.     

The effect of cyclo-DfKRG peptide immobilization on titanium on the adhesion and differentiation of human osteoprogenitor cells

This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated. Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.     

Electropolymerization of dopamine for surface modification of complex-shaped cardiovascular stents

Inspired by the adhesion strategy of marine mussels, self-polymerization of dopamine under alkaline condition has been proven to be a simple and effective method for surface modification of biomaterials. However, this method still has many drawbacks, such as the use of alkaline aqueous medium, low poly(dopamine) deposition rate, and inefficient utilization of dopamine, which greatly hinder its practical application. In the present study, we demonstrate that electropolymerization of dopamine is a facile and versatile approach to surface tailoring of metallic cardiovascular stents, such as small and complex-shaped coronary stent. Electropolymerization of dopamine leads to the formation of a continuous and smooth electropolymerized poly(dopamine) (ePDA) coating on the substrate surface. This electrochemical method exhibits a higher deposition rate and is more efficient in dopamine utilization compared with the typical self-polymerization method. The ePDA coating facilitates the immobilization of biomolecules onto substrates to engineer biomimetic microenvironments. In vitro and in vivo experiments demonstrate that ePDA coating functionalized with vascular endothelial growth factor can greatly enhance the desired cellular responses of endothelial cells and prevent the neointima formation after stent implantation. The proposed methodology may find applications in the area of metallic surface engineering, especially for the cardiovascular stents and potentially all biomedical devices with electroconductive surface as well.     

The interplay between inflammasome activation and antifungal host defense

Fungal infections cause significant morbidity and mortality in humans, and they are a growing problem due to the increased usage of broad-spectrum antibiotics and immunosuppressive therapies. The equilibrium between the commensal microbial flora and the immune system that protects the host against invasive fungal infection is disturbed during disease, and understanding this disturbed balance is important to develop new therapeutic interventions for the treatment of fungal infection. In the context of tolerating fungi during colonization and eliciting a vigorous immune response to eliminate invading fungal pathogens when needed, the inflammasome has been identified as an integral component of antifungal host defense. It contributes to mucosal host defense by regulating T-helper 17 (Th17) cell responses, and contributes to protective responses such as neutrophil influx during fungal sepsis. Several aspects are important for understanding the role of the inflammasome for antifungal host defense, such as the role of fungal cell wall morphology and its components in triggering the inflammasome, the pattern recognition pathways and downstream signaling cascades involved in the activation of the inflammasome, and the effects of inflammasome activation during fungal infection. The future perspectives of inflammasome research in fungal immunology, with emphasis on targeting the inflammasome for the design of future immunotherapies, is also discussed.     

In vitro antibacterial and cytotoxicity assay of multilayered polyelectrolyte-functionalized stainless steel

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic and implant surgery. In the present study, a new strategy for confering stainless steel with antibacterial property via the alternate deposition of quaternized polyethylenimine (PEI) or quaternized polyethylenimine-silver complex and poly(acrylic acid) (PAA) was investigated. The success of the deposition of the polyelectrolyte multilayers (PEM) and its chemical nature was investigated by static water contact angle and X-ray photoelectron spectroscopy (XPS), respectively. The antibacterial activity was assessed using Escherichia coli (E. coli, a gram-negative bacterium) and Staphylococcus aureus (S. aureus, a gram-positive bacterium). The inhibition of E. coli and S aureus growth on the surface of functionalized films was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy. The cytotoxicity of the PEM to mammalian cells, evaluated by the MTT assay, was shown to be minimal and long-term antibacterial efficacy can be maintained. These results indicate new possibilities for the use of such easily built and functionalized architectures for the functionalization of surfaces of implanted medical devices.     

Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-silver ions complex

Antibacterial multilayer films containing nanosilver were prepared via layer-by-layer fashion. PET film was aminolyzed with 1,6-hexanediamine to introduce amino groups on PET film surface; chitosan-silver nitrate complex and heparin were alternately deposited onto an aminolyzed PET film surface, and subsequently, the silver ions within the multilayer films were reduced with ascorbic acid to form silver nanoparticles. UV-visible spectroscopy and transmission electron microscopy confirmed the formation of well-dispersed nanosilver particles with sizes (10-40 nm) that depended on the initial concentration of silver ions in chitosan solution and the pH of ascorbic acid solution. The chitosan/heparin multilayer films were possessed of bactericidal effect on Escherichia coli (E. coli), and this antibacterial effect could be significantly enhanced by the incorporation of silver nanoparticles into the multilayer films. The multilayer films containing nanosilver were not only effective as antibacterial but also as anticoagulant coating. And cell toxicity evaluation suggested that the multilayer films containing nanosilver did not show any cytotoxicity. The multilayer films containing nanosilver may have good potentials for surface modification of medical devices, especially for cardiovascular implants.     

Covalent functionalization of NiTi surfaces with bioactive peptide amphiphile nanofibers

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium (NiTi) shape memory alloy in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO(2) layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO(2) with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM techniques. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers.     

Spatial control of cellular adhesion using photo-crosslinked micropatterned polyelectrolyte multilayer films

Cellular patterning on biomaterial surfaces is important in fundamental studies of cell–cell and cell–substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. In this study, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithographic technique to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. This technique can be also applied to other PEM systems with proper rinsing protocol, and many types of substrates. Cell co-culture systems can be also achieved by this technique.     

Regulation of fungal infection by a combination of amphotericin B and peptide 2, a lactoferrin peptide that activates neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 x 10(8) cells) or Aspergillus fumigatus (1 x 10(8) cells) was prolonged 3 to 5 days by the injection of 10 microg of peptide 2 (a lactoferrin peptide) and 10 microg of alpha-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 microg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 microg of amphotericin B. When mice received a combined injection of peptide 2 (10 microg/day) with amphotericin B (0.5 microg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as alpha-defensin 1, beta-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O2-) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O2- -generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47phox as well as p67phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565?±?20?ng/cm(2) of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I(2) (PGI(2)). In?vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents.     

Coating polyvinylchloride surface for improved antifouling property

Antifouling surfaces are specifically crucial to cardiovascular applications. In this study, a polyvinylchloride (PVC) surface was modified by coating a biocompatible and hydrophilic polymer by a mild coating technique. The PVC surface was first activated and then functionalized, followed by coating with the polymer. Results show that the coated hydrophilic polymer significantly reduced 3T3 fibroblast cell adhesion as well as bacteria adhesion. The 3T3 cell adhesion to the polymer-coated surface was reduced to 52–66% as compared to the original PVC surface. Bacterial adhesion to the polymer-coated surface was reduced to 61–80% for Pseudomonas aeruginosa, 65–81% for Staphylococcus aureus, and 73–85% for Escherichia coli, as compared to the original PVC surface. It appears that this novel polymer-coated PVC surface has an antifouling property.     

The Exciting Future of Antifungal Therapy

 Invasive fungal infections are becoming more common. Current therapy is generally limited to amphotericin B in its parent and lipid formulations, 5-fluctyosine, fluconazole, and itraconazole. Toxicity, drug-drug interactions, and increasing fungal resistance reduce the usefulness of these drugs, and the need for new therapies is pressing. This article briefly discusses the limitations of antifungal minimum inhibitory testing, and then summarizes new antifungal drugs in development that have been tested in humans. It also addresses novel treatment strategies such as drug combination therapy, pharmacological reformulations to improve the efficacy or reduce the toxicity of current antifungal drugs, immune function augmentation, and vaccine development. All of these strategies, although in their infancy, will enhance the clinician's ability to care for patients with invasive fungal infections.     

Functionalized PEG hydrogels through reactive dip-coating for the formation of immunoactive barriers

Influencing the host immune system via implantable cell-delivery devices has the potential to reduce inflammation at the transplant site and increase the likelihood of tissue acceptance. Towards this goal, an enzymatically-initiated, dip-coating technique is adapted to fabricate conformal hydrogel layers and to create immunoactive polymer coatings on cell-laden poly(ethylene glycol) (PEG) hydrogels. Glucose oxidase (GOx)-initiated dip coatings enable the rapid formation of uniform, PEG-based coatings on the surfaces of PEG hydrogels, with thicknesses up to 500 μm where the thickness is proportional to the reaction time. Biofunctional coatings were fabricated by thiolating biomolecules that were subsequently covalently incorporated into the coating layer via thiol-acrylate copolymerization. The presence of these proteins was verified via fluorescent confocal microscopy and a modified ELISA, which indicated IgG concentrations as high as 13 ± 1 ng/coated cm2 were achievable. Anti-Fas antibody, known to induce T cell apoptosis, was incorporated into coatings, with or without the addition of ICAM-1 to promote T cell interaction with the functionalized coating. Jurkat T cells were seeded atop functionalized coatings and the induction of apoptosis was measured as an indicator of coating bioactivity. After 48 h of interaction with the functionalized coatings, 61 ± 9% of all cells were either apoptotic or dead, compared to only 18 ± 5% of T cells on non-functionalized coatings. Finally, the cytocompatibility of the surface-initiated GOx coating process was confirmed by modifying gels with either encapsulated β-cells or 3T3 fibroblasts within a gel that contained a PEG methacrylate coating.     

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.