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).

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.

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.

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.

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.

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.

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.

Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications.

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.

Interaction between proteins and polyphosphazene derivatives having a galactose moiety.

For tissue engineering applications, it is necessary to balance the need for specific biological interactions with the need to prevent unfavorable nonspecific interactions. For this purpose, novel poly[(organo)phosphazenes] were synthesized having galactose and/or poly(ethylene glycol) (PEG) side chains. The synthesis was described previously. Here, we investigate the human serum albumin (HSA) adhesion to these polymers using surface plasmon resonance (SPR). We could conclude that the incorporation of PEG reduced the protein adsorption. The influence of the galactose moieties was investigated using SPR and a sugar-lectin binding assay. The interaction between a lectin (Peanut agglutinin, PNA or Ricinus communis-agglutinin, RCA) and the polyphosphazene derivatives was evaluated. Type IIA polymers, having aminohexyl-galactose, phenylalanine ethyl ester, and glycine ethyl ester side chains, were capable of binding with the lectin. As the amount of galactose was increased, the extent of the galactose specific lectin binding was also increased (higher RU or absorbance). PEG containing polymers failed to bind specifically with the lectin. The presence of PEG, either as a spacer or as additional chains, interfered with the establishment of contact between the galactose and the binding site on the lectin. The adsorption of PNA or RCA to these types of polymers was attributed to nonspecific interactions. SPR was also used to determine rate and equilibrium constants. In addition the effect of the addition of water soluble polyphosphazenes on the enzymatic cleavage of o-nitrophenyl-beta-D-galactopyranoside was investigated. The galactose moieties were not available as inhibitors because of the presence of PEG.

Evaluation of an injectable, photopolymerizable three-dimensional scaffold based on D: ,L: -lactide and epsilon-caprolactone in a tibial goat model.

An in situ crosslinkable, biodegradable, methacrylate-encapped porous bone scaffold composed of D: ,L: -lactide, epsilon-caprolactone, 1,6-hexanediol and poly(ortho-esters), in which crosslinkage is achieved by photoinitiators, was developed for bone tissue regeneration. Three different polymer mixtures (pure polymer and 30% bioactive glass or alpha-tricalcium phosphate added) were tested in a uni-cortical tibial defect model in eight goats. The polymers were randomly applicated in one of four (6.0 mm diameter) defects leaving a fourth defect unfilled. Biocompatibility and bone healing properties were evaluated by serial radiographies, histology and histomorphometry. The pure polymer clearly showed excellent biocompatibility and moderate osteoconductive properties. The addition of alpha-TCP increased the latter characteristics. This product offers potentials as a carrier for bone healing promoter substances.

Immediate postextraction implant placement in sheep's mandibles: a pilot study.

PURPOSE: In this study, sheep were examined as a potential animal model for immediate implant placement in fresh extraction sockets using experimental photopolymerisable bioabsorbable polymers. MATERIALS: A total of 22 cylindrical implants were placed in fresh mandibular premolar extraction sockets of 7 sheep. Residual bone-implant voids were filled with a biocompatible composite of poly-methyl-methacrylate and poly-hydroxyl-ethyl-methacrylate (Bioplant 24). Photopolymerisation of a viscose mixture of experimental prepolymers and Bioplant 24 applied to the neck of the implants provided additional support before gingival closure. Clinical and radiographic controls were performed 30, 90, and 180 days after surgery. At 180 days postoperatively, the sheep were sacrificed and the mandibular segments were isolated for histological processing. RESULTS: High cumulative implant failure rates of 45.5%, 63.6%, and 77.3% at respectively 30, 90 and 180 days were recorded. Significantly more implants were lost when the position of the neck was located above the level of the alveolar crest (P < 0.05). Clinical and histological observations demonstrated poor implant osseointegration characterized by ingrowth of soft tissue into the extraction sockets. Bone substitutes were lost in all cases. DISCUSSION AND CONCLUSION: Sheep have many practical advantages compared with other animal models. However, their specific oral biomechanics inherent to their constant ruminant activity accounted for a high degree of the reported implant failures. Important adaptations to the implantation technique and postoperative management will be necessary to use sheep as an animal model for future oral implant related experiments.

Silicon-on-Insulator microring resonator for sensitive and label-free biosensing.

Label-free biosensors attempt to overcome the stability and reliability problems of biosensors relying on the detection of labeled molecules. We propose a label-free biosensor based on microring cavities in Silicon-on-Insulator (SOI) that fits in an area below 10x10mum(2). The resonance wavelength shift that occurs when the surroundings of a cavity is changed, is used for sensing. While theoretically the performance for bulk refractive index changes is moderate (10(-5)), this device performs outstanding in terms of absolute molecular mass sensing (theoretical sensitivity of 1fg molecular mass) thanks to its extremely small dimensions. We use the avidin/biotin high affinity couple to demonstrate good repeatability and detection of protein concentrations down to 10ng/ml. Fabrication with Deep UV lithography allows for cheap mass production and integration with electronic functions for complete lab-on-chip devices.

Polyacetal and poly(ortho ester)-poly(ethylene glycol) graft copolymer thermogels: preparation, hydrolysis and FITC-BSA release studies.

Graft copolymers comprised of a polyacetal backbone with pendant poly(ethylene glycol) side-chains were prepared using a condensation reaction between a divinyl ethers, a diol and Fmoc-protected serinol, followed by deprotecting the amine and reacting the polyacetal with pendant amino groups with PEG-alpha-methoxy-omega-succimidylcarbonate. A series of materials having lower critical solution temperature (LCST) between 25 and 60 degrees C has been prepared. Since LCST is determined by the hydrophilic-hydrophobic balance, and this in turn is determined by the molecular weight of the polyacetal backbone, the molecular weight of the grafted PEG and the amount grafted, materials having a desired LCST could be readily prepared. Incorporating FITC-BSA at 1 wt.% into the thermogel resulted in sustained release over about 100 days at pH 7.4 and 40 days at pH 5.5 without a burst and by reasonably linear kinetics. Incorporating FITC-BSA at 5 wt.% into the thermogel significantly increased delivery time at pH 5.5 and decreased the difference in delivery rates between pH 5.5 and pH 7.4. FITC-BSA is released by a predominantly erosion-controlled process and FITC-BSA depletion coincides closely with total gel dissolution. More rapidly eroding thermogels were prepared by replacing the polyacetal backbone with a poly(ortho ester) backbone. Such gels completely dissolved between 3 and 6 days. It is hoped that intermediate erosion rates can be achieved by preparing backbones containing both acetal and ortho ester linkages. Such materials have been prepared and shown to have LCST values in the desired range, but no erosion, or drug release studies have as yet been completed.

Vinyl polymers as non-viral gene delivery carriers: current status and prospects.

Since the first application of polymers as non-viral gene delivery systems in 1965 by Vaheri and Pagano using functionalised dextran (A. Vaheri and J. S. Pagano, "Infectious poliovirus RNA: a sensitive method of assay", Virology 1965, 27, 434-6), a large number of different polymers have been developed, studied and compared for application as DNA carriers. Vinyl-based polymers are one type of polymers that have gained considerable interest. The interest in developing this particular type of polymer is partly related to the straightforward way in which large amounts of these polymers can be prepared by radical (co)polymerisation. This opens up a path for establishing a wide range of structure-property relations using polymer libraries. The present review aims to give an overview of past and ongoing research using vinyl-based gene delivery systems. The application of cationic, neutral and zwitterionic polymers as DNA carriers is summarised and discussed. [structure: see text] Chemical structure of DEAE-functionalised dextran.

Improved anti-inflammatory properties for naproxen with cyclodextrin-grafted polysaccharides.

Mannan and carboxymethylcellulose, previously activated by periodate oxidation, were grafted with mono-6-butylenediamino-6-deoxy-beta-cyclodextrin derivatives by reductive alkylation in the presence of sodium borohydride. The formation of supramolecular complexes between these polymers and Naproxen was confirmed by fluorescence spectroscopy. The solubility of the drug was 3.8-4.6 fold increased in the presence of the cyclodextrin-grafted polysaccharides. The in vivo anti-inflammatory property of Naproxen was 1.7 times higher after supramolecular association with beta-cyclodextrin-branched mannan.

Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures.

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.

Tailor-made polymers for local drug delivery: release of macromolecular model drugs from biodegradable hydrogels based on poly(ethylene oxide).

Hydrogels were synthesized from degradable and non-degradable PEO bismacromonomers. The degradability was provided by hydrolyzable segment between the main PEO chain and the methacrylate or methacrylamide groups at the both PEO chain termini. The hydrolyzable segment consisted of a monomeric alpha-hydroxy acid or a depsipeptide. The polymerization conditions and the choice of a bismacromonomer influenced the cross-linking density of the hydrogels. The release behavior of model macromolecular solutes, FITC dextran and bovine serum albumin (BSA), was studied. The small FITC-dextran 4 kDa was released rapidly from the hydrogel. The larger FITC-dextran 40 kDa and BSA were retained inside the matrix and their release rate was controlled by the degradation.

Synthesis and in vitro evaluation of macromolecular antitumour derivatives based on phenylenediamine mustard.

Poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG) and poly(ethylene glycol) (PEG)-grafted PHEG conjugates of N,N-di(2-chloroethyl)-4-phenylenediamine mustard (PDM) were synthetised. A collagenase-sensitive oligopeptide spacer was selected to link the cytotoxic agent PDM onto the polymeric carrier. First, the oligopeptide-drug conjugate, L-pro-L-leu-gly-L-pro-gly-PDM, was prepared. In a second step, the low molecular weight PDM derivative and PEG-NH(2) were coupled to a N,N-disuccinimidylcarbonate activated PHEG. Dynamic laser light scattering measurements indicated the formation of aggregates. The presence of human serum albumin had no significant effect on the diameter of the conjugates. The hydrolytic stability of the conjugates was investigated in buffer solutions. The conjugates showed an improved stability compared to the parent nitrogen mustard. The enzymatic degradation studies of the polymeric conjugates were performed in the presence of collagenase type IV (Clostridiopeptidase A; EC 3.4.24.3), cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5) and tritosomes. Only the bacterial collagenase type IV was able to cleave the spacer releasing free PDM and its peptidyl derivative, gly-L-pro-gly-PDM. The in vitro cytotoxicity of the conjugates was evaluated against HT1080 fibrosarcoma cells and MDA adenocarcinoma cells. All conjugates showed low toxicity towards these cell lines.

Enhancement of polymethacrylate-mediated gene delivery by Penetratin.

Polymethacrylates are vinyl-based polymers that are used for DNA transfection. Cationic polymethacrylates efficiently condense DNA by forming inter-polyelectrolyte complexes. Their use for DNA transfection is, however, limited due to their low ability to interact with membranes. In order to increase their transfection efficiency, we combined polymethacrylates with Penetratin, a 16-residue water-soluble peptide that internalises into cells through membrane translocation. DNA condensation was assessed using physicochemical methods, while transfection efficiency and cellular internalisation were studied using Cos-1 cells. Agarose gel electrophoresis retardation, ethidium bromide exclusion tests and dynamic light scattering measurements showed that the stability of the polymethacrylate-DNA complexes is not affected by addition of Penetratin. Transfection efficiency of polymethacrylate-DNA complexes into Cos-1 cells increased by addition of Penetratin and was higher than that of polyethylenimine (PEI)-DNA complexes and comparable to Lipofectamine. Confocal microscopy and flow cytometry indicated that Penetratin mainly enhances endolysosomal escape polymethacrylate-DNA complexes and increases their cellular uptake. Since the cellular toxicity of polymethacrylate-DNA-Penetratin complexes remains low, especially compared to PEI, this transfection system opens new perspectives for gene therapy.

Improved anti-inflammatory and pharmacokinetic properties for superoxide dismutase by chemical glycosidation with carboxymethylchitin.

O-carboxymethylchitin (molecular weight = 1.07 x 10(5), degree of carboxymethylation = 80%, degree of N-acetylation = 91%) was chemically attached to superoxide dismutase by the formation of amide linkages through a carbodiimide catalyzed reaction. The glycosidated enzyme contained about 1.8 mole of polysaccharide per mole of protein and retained 57% of the initial catalytic activity. The anti-inflammatory activity of the enzyme was 2.4 times increased after conjugation with the polysaccharide. The modified superoxide dismutase preparation was remarkably more resistant to inactivation with H(2)O(2) and its plasma half-life time was prolonged from 4.8 min to 69 h.