Immunocompatibility evaluation of hydrogel-coated polyimide implants for applications in regenerative medicine.

Immunocompatibility of gelatin-based hydrogels to be applied as implant coatings for local regenerative treatment has been studied. First, the bio- and immuno-acceptability of the methacrylamide-modified gelatin hydrogels per se was screened. The results indicated that the hydrogels support cell growth. Metabolic activity of normal cells and permanent cell lines representing various cell types (endothelial, epithelial, fibroblast, and monocyte/macrophage) cultivated on the gelatin hydrogels was moderately lower compared to cells cultivated on tissue culture plastic. The cells cultivated on the hydrogels produced identical cytokines as the control cells although at lower levels. Importantly, no inflammatory activity, measured by nitric oxide and pro-inflammatory cytokine (IL-1α, IL-6, and TNFα) production, was observed in peritoneal cells and monocyte/macrophage RAW 264.7 cell line cultivated on the hydrogels. Finally, polyimide (PI) implantable membranes were surface-modified with gelatin hydrogels and screened for their in vivo immunocompatibility. Their histological examination performed after subcutaneous implantation in mice produced a sound proof of immunoacceptability. Normal tissue repair, mild cellular infiltration and edema mainly induced by the surgery were observed after 2 and 6 days. No adverse tissue responses were induced by the implants. Analysis performed after 4 and 9 weeks indicated areas of foreign body granuloma without formation of a fibrous capsule.

Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications.

An electronic sensor system for urinary bladder pressure monitoring requires an imbedding into a biocompatible, flexible, and liquid-impermeable material. Poly(dimethylsiloxane) (PDMS) was selected in the present set-up as packaging material because it fulfills the abovementioned requirements. However, the surface of PDMS is hydrophobic and causes undesired interactions with salts, proteins, and cells present in urine. To reduce possible interactions of urine salts in the urinary bladder, monomers, [2-(methacryloyloxy)ethyl]-dimethyl-3-sulfopropyl-ammonium hydroxide (sulfobetaine) and 2-acrylamido-2-methylpropyl sulfonic acid, were grafted onto the surface through oxygen plasma treatment. A reduction in salt deposition between the pure PDMS and the modified PDMS was observed both in vitro (artificial urine flow over the surface) and in vivo (implants into the urinary bladder of experimental pigs). Additionally, a 10-fold reduction in salt deposition was observed in vitro due to grafting of the monomers onto the surface. These modified PDMS materials proved also to be biocompatible in cell cultures, which was further confirmed by histological screening of the bladder tissue after implantation in an in vivo pig model.

Cell survival and proliferation after encapsulation in a chemically modified Pluronic(R) F127 hydrogel.

Pluronic® F127 is a biocompatible, injectable, and thermoresponsive polymer with promising biomedical applications. In this study, a chemically modified form, i.e., Pluronic ALA-L with tailored degradation rate, was tested as an encapsulation vehicle for osteoblastic cells. UV cross-linking of the modified polymer results in a stable hydrogel with a slower degradation rate. Toxicological screening showed no adverse effects of the modified Pluronic ALA-L on the cell viability. Moreover, high viability of embedded cells in the cross-linked Pluronic ALA-L was observed with life/death fluorescent staining during a 7-day-culture period. Cells were also cultured on macroporous, cross-linked gelatin microbeads, called CultiSpher-S® carriers, and encapsulated into the modified cross-linked hydrogel. Also, in this situation, good cell proliferation and migration could be observed in vitro. Preliminary in vivo tests have shown the formation of new bone starting from the injected pre-loaded CultiSpher-S® carriers.

From polyesters to polyamides via O-N acyl migration: an original multi-transfer reaction.

A new strategy for the synthesis of polyamides from polyesters of hydroxyl-containing amino acids using a multi O-N acyl transfer reaction was developed. This original approach allowed the synthesis of three generations of polymers from the same starting monomer. The polymerization of N-benzyloxycarbonyl-serine and its γ-homologated derivative provided the Z-protected polyesters; then the water-soluble polycationic polyesters were obtained by removal of the Z-protecting group; and finally the polyamides were obtained by a base-induced multi O-N acyl transfer, both in aqueous or organic medium. The key step transfer reaction was monitored by the disappearance and appearance of characteristic NMR proton signals and IR bands of polyesters and polyamides.

Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 µm), highly porous (Ø 1 µm spaced 3 µm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes.

There is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Such methods are urgently needed to support the optimisation of interaction of substances, particles or materials with cell. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer-DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed. It was shown for the first time that the impedance-based method predicated on the microhole chip can be used for biological relevant substances at relevant concentrations and that it is more sensitive than the well-established biological marker.

Design of an imprinted clean-up method for mycophenolic acid in maize.

In the present work, the development of imprinted polymers selective towards mycophenolic acid and their application in food analysis are reported for the first time. To synthesize the molecularly imprinted polymer (MIP) 4-vinylpyridine and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Besides the toxin itself, the implementation of structural analogues as templates was evaluated. A molecularly imprinted solid-phase extraction (MISPE) procedure was designed for the selective clean-up of maize extracts. Binding experiments and Scatchard analysis indicated the presence of specific binding sites in the imprinted polymers. The imprinting effect varied along with the selected template. The dissociation constant (K(D)) of the higher affinity binding sites ranged from 0.8 µmol/l to 15.6 µmol/l, while the K(D) of the lower affinity binding sites was in the range of 138.5-519.3 µmol/l. The performance of the MIPs throughout the clean-up of spiked maize sample extracts was evaluated and compared with the results obtained when applying a non-imprinted polymer. Depending on the polymers and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 49% to 84% and from 28% to 31%. The imprinted polymers were superior regarding matrix effect, limit of detection (LOD) and limit of quantification (LOQ). LOD ranged from 0.17 µg/kg to 0.25 µg/kg and LOQ varied from 0.57 µg/kg to 0.82 µg/kg. Analysis of 15 maize samples by liquid chromatography tandem mass spectrometry revealed that the MIPs could be excellent sorbents for clean-up of contaminated food samples.

Plasma-induced surface modification of polydimethylsiloxane aimed at reducing salt and protein deposition.

Polydimethylsiloxane (PDMS) is an elastomer that is widely used in construction and for biological and biomedical applications. The biocompatibility of PDMS was improved by different surface treatment methods, i.e., plasma treatment or a combination of plasma treatment with UV-irradiation or redox initiator, to minimize the effects of deposition of salts and proteins. In this work we used the vinyl monomers sulfobetaine and AMPS which have good biocompatible properties.

Static secondary ion mass spectrometry for the surface characterisation of individual nanofibres of polycaprolactone functionalised with an antibacterial additive.

Electrospinning (ES) of polymer solutions generates non-woven webs of nanofibres. The fibre diameter ranges between 10 nm and 1 µm depending on the operating conditions. Surface functionalisation can be performed by the use of suitable additives. Detailed characterisation of the molecular composition at the fibre surface is a key issue. Biodegradable nanowebs with potential antibacterial activity have been prepared by ES of solutions containing polycaprolactone (PCL) and a functionalising additive with PCL segments and hexyldimethylammonium groups (PCLhexaq). Static secondary ion mass spectrometry with Bi(3)(+) projectiles has been applied to individual nanofibres. The positive ion mass spectra contain several signals with high structural specificity allowing the presence of PCLhexaq to be traced back in spite of its low concentration (0.16-1.4% w/w relative to PCL) and its structural similarity to the PCL fibre matrix. Imaging of structural ions visualises the homogeneous distribution of PCLhexaq over the fibre surface. Quantifying the surface concentration of PCLhexaq relative to that of PCL reveals electric field-driven surface enrichment of the additive during ES. Finally, nanofibres subjected to leaching in water for up to 72 h have been analysed. The PCLhexaq surface concentration decreases almost linearly with time at a rate of 0.6% h(-1).

Post-plasma grafting of AEMA as a versatile tool to biofunctionalise polyesters for tissue engineering.

In the last decade, substantial research in the field of post-plasma grafting surface modification has focussed on the introduction of carboxylic acids on surfaces by grafting acrylic acid (AAc). In the present work, we report on an alternative approach for biomaterial surface functionalisation. Thin poly-ε-caprolactone (PCL) films were subjected to a dielectric barrier discharge Ar-plasma followed by the grafting of 2-aminoethyl methacrylate (AEMA) under UV-irradiation. X-ray photoelectron spectroscopy (XPS) confirmed the presence of nitrogen. The ninhydrin assay demonstrated, both quantitatively and qualitatively, the presence of free amines on the surface. Confocal fluorescence microscopy (CFM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise the grafted surfaces, indicating the presence of pAEMA. Static contact angle (SCA) measurements indicated a permanent increase in hydrophilicity. Furthermore, the AEMA grafted surfaces were applied for comparing the physisorption and covalent immobilisation of gelatin. CFM demonstrated that only the covalent immobilisation lead to a complete coverage of the surface. Those gelatin-coated surfaces obtained were further coated using fibronectin. Osteosarcoma cells demonstrated better cell-adhesion and cell-viability on the modified surfaces, compared to the pure PCL films.

Prevention of Candida albicans biofilm formation by covalently bound dimethylaminoethylmethacrylate and polyethylenimine.

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.

Synthesis and application of a T-2 toxin imprinted polymer.

The synthesis of a T-2 toxin imprinted polymer and its application in food analysis are reported for the first time. A molecularly imprinted polymer (MIP) for the selective recognition of T-2 toxin (T-2) was synthesized by bulk polymerization. Methacrylamide and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Molecularly imprinted solid-phase extraction (MISPE) procedures were optimized for further application in the analysis of T-2. Scatchard plot analysis revealed that two classes of imprinted binding sites were formed in the imprinted polymer. The dissociation constant (KD) of the higher affinity binding sites was 7.0 micromol/l, while the KD of the lower affinity binding sites was 54.7 micromol/l. The performance of the MIP throughout the clean-up of spiked maize, barley and oat sample extracts was compared with the results obtained when using non-imprinted polymer, OASIS HLB and immunoaffinity columns (IAC). Depending on the food matrix and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 60% to 73% and from 21% to 57%. Recoveries obtained after clean-up using OASIS HLB and IAC were in the range of 74-104% and 60-85%, respectively. Although highest recoveries were obtained with OASIS HLB sorbents, the designed MIP and the IAC were superior regarding selectivity, cross-reactivity, matrix effect, limits of detection (LOD) and limits of quantification (LOQ). Depending on the matrix, LOD after MISPE ranged from 0.4 microg/kg to 0.6 microg/kg and LOQ from 1.4 microg/kg to 1.9 microg/kg. LOD and LOQ after OASIS HLB clean-up varied from 0.9 microg/kg to 3.5 microg/kg and from 3.1 microg/kg to 11.7 microg/kg, respectively. The LOD and LOQ values obtained with IAC were in the range of 0.3-2.3 microg/kg and 1.0-7.7 microg/kg, respectively. Analysis of 39 naturally contaminated samples (maize, barley and oat) by liquid chromatography tandem mass spectrometry revealed that the MIP could be an excellent alternative for clean-up of contaminated food samples.

Candida albicans biofilm formation on peptide functionalized polydimethylsiloxane.

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.

Inhibition of Candida albicans biofilm formation by antimycotics released from modified polydimethyl siloxane.

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.

Self-hardening calcium deficient hydroxyapatite/gelatine foams for bone regeneration.

In this work gelatine was used as multifunctional additive to obtain injectable self-setting hydroxyapatite/gelatine composite foams for bone regeneration. The foaming and colloidal stabilization properties of gelatine are well known in food and pharmaceutical applications. Solid foams were obtained by foaming liquid gelatine solutions at 50 degrees C, followed by mixing them with a cement powder consisting of alpha tricalcium phosphate. Gelatine addition improved the cohesion and injectability of the cement paste. After setting the foamed paste transformed into a calcium deficient hydroxyapatite. The final porosity, pore interconnectivity and pore size were modulated by modifying the gelatine content in the liquid phase.

Evaluation of bone regeneration with an injectable, in situ polymerizable Pluronic F127 hydrogel derivative combined with autologous mesenchymal stem cells in a goat tibia defect model.

In situ forming bone substitute materials are attractive candidates for filling irregularly shaped defects. In this study, a chemically modified form of the Pluronic F127 hydrogel was used. Similar to the parent form, this derivative underwent a sol-gel transition in the body and additional radical curing resulted in a stable three-dimensional network gel with a controllable degradation rate. An extra cell source of autologous bone marrow-derived mesenchymal stem cells was mixed with the hydrogel to increase the ossification process, when implanted in noncritical size unicortical tibia defects. These cells were cultured and predifferentiated on two types of cell carrier systems, that is, gelatin CultiSpher-S microcarriers and hydroxyapatite tubular carriers. Radiographic and histological evaluation revealed that bone regeneration was comparable in the defects with the bone substitute compositions and the untreated control defects at 2 and 4 weeks postimplantation and that newly formed bone originated from the cells on the CultiSpher-S carriers. This resulted, 6 and 8 weeks postimplantation, in faster bone repair in the defects filled with the hydrogel plus CultiSpher-S carriers in comparison to the control defects. Surprisingly, there was no formation of new bone originating from the hydroxyapatite carriers. The hydrogel by itself seemed to stimulate the natural repair process.

In vitro cytotoxicity testing and the application of elastic interconnection technology for short-term implantable electronics.

An electronic device was fabricated consisting of 2 flexible electronic circuit islands, interconnected by a 7 cm long elastic interconnection, which could be elongated for at least 50%. This interconnection was based on gold conductor tracks following a 2-D spring pattern, embedded in a biocompatible silicone elastomer. The complete device was embedded in the same silicone elastomer. An in vitro cytotoxicity extraction test, executed on small test-samples in accordance with the ISO 10 993-1 guidelines, revealed that the applied silicone encapsulation to these samples functioned as a good seal for at least 8 days.

Modification of polydimethylsiloxane surfaces using benzophenone.

New biocompatible materials have been obtained by different modifications of polydimethylsiloxane (PDMS) surfaces. PDMS is of great interest for several biomedical applications. For some applications the native silicone does not provide an optimal performance. PDMS attracts proteins and salts. To reduce protein adhesion and salt deposition selected monomers were grafted by radical polymerization on the silicone surface. The conditions for surface modifications of PDMS using benzophenone as UV initiator were optimized. The modified surfaces were characterized properly using different methods. The effect of surface modifications on the albumin, as model protein, deposition was tested in an in vitro model.

Nonthermal plasma technology as a versatile strategy for polymeric biomaterials surface modification: a review.

In modern technology, there is a constant need to solve very complex problems and to fine-tune existing solutions. This is definitely the case in modern medicine with emerging fields such as regenerative medicine and tissue engineering. The problems, which are studied in these fields, set very high demands on the applied materials. In most cases, it is impossible to find a single material that meets all demands such as biocompatibility, mechanical strength, biodegradability (if required), and promotion of cell-adhesion, proliferation, and differentiation. A common strategy to circumvent this problem is the application of composite materials, which combine the properties of the different constituents. Another possible strategy is to selectively modify the surface of a material using different modification techniques. In the past decade, the use of nonthermal plasmas for selective surface modification has been a rapidly growing research field. This will be the highlight of this review. In a first part of this paper, a general introduction in the field of surface engineering will be given. Thereafter, we will focus on plasma-based strategies for surface modification. The purpose of the present review is twofold. First, we wish to provide a tutorial-type review that allows a fast introduction for researchers into the field. Second, we aim to give a comprehensive overview of recent work on surface modification of polymeric biomaterials, with a focus on plasma-based strategies. Some recent trends will be exemplified. On the basis of this literature study, we will conclude with some future trends for research.

Affinity study of novel gelatin cell carriers for fibronectin.

In the present work, the gelatin/fibronectin affinity was evaluated using SPR, QCM and radiolabelling. The results indicate that type A gelatin films possess a higher affinity for Fn compared to type B gelatin. This is due to a combined hydrophobic and electrostatic interaction between gelatin type A and Fn. In a second part, the affinity of Fn for porous gelatin scaffolds was evaluated. The scaffolds were prepared by a cryogenic treatment and subsequent freeze-drying yielding type I and type II scaffolds which possess different pore geometries/sizes. The results indicate that the Fn density on the scaffolds can be fine-tuned by varying the Fn concentration, the gelatin type (A vs. B), the pore size/geometry (type I vs. type II scaffolds).