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.

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.

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.

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.

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.

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.

SOI optical microring resonator with poly(ethylene glycol) polymer brush for label-free biosensor applications.

Label-free monitoring of biomolecular interactions has become of key importance for the emerging proteomics field. Monitoring real time interaction kinetics and high throughput screening of complex samples is of major importance for a variety of applications. We previously reported the use of Silicon-on-Insulator photonics microring resonators for cheap disposable biosensors on chip. Silicon photonics is a platform for micro- and nanoscale integrated devices that can be fabricated at extremely low cost, with standard CMOS processing facilities. Incorporation of a hydrophilic heterobifunctional polymer coating on the silicon chips largely improved the system's response to non-specific binding. We report the chemical coating procedure, the chemical surface characterization and optical measurements for both specific and non-specific interactions. Two heterobifunctional polymer coatings were investigated, alpha-sulfanyl-omega-carboxy-poly(ethylene glycol) and monoprotected diamino-poly(ethylene glycol). Homogenous coatings with thicknesses of 2.3 and 2.5 nm were obtained, corresponding to a surface loading of 99 pm/cm(2) carboxy- and 97 pm/cm(2) aminogroups, respectively. The polymer coated sensor with covalently bound biotin receptor molecules showed very low response to Bovine Serum Albumin (BSA) up to 1 mg/ml in contrast to a high response to avidin with much lower concentrations (2, 10, 87.5 and 175 microg/ml). By extrapolation the detection limit is about 10 ng/ml or 0.37 fg avidin mass. Comparison with the values reported for standard silanization confirms the polymer coating does not deteriorate the system's limit of detection. This makes the optical biosensor chip suitable to be integrated in a microflow system for commercial label-free biosensors and for lab-on-a-chip applications.

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.

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.

Synthesis and characterization of novel poly[(organo)phosphazenes] with cell-adhesive side groups.

There is a need to develop new scaffold materials with controlled surface properties for tissue engineering applications. For that purpose novel biodegradable poly[(organo)phosphazenes] were synthesized. A cell-binding molecule, galactose, was introduced via a spacer, either 6-aminohexanol (AH) or poly(ethylene glycol) (PEG; M(w) = 3400). Some polymers were substituted with an additional PEG chain of different molecular weights (M(w) = 750 or 5000). The polyphosphazene derivatives were characterized by 1H NMR. T(g) and T(m) were determined using differential scanning calorimetry. A detailed surface analysis of the polymers using X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS), and dynamic contact angle (DCA) measurements was performed. Typical backbone and side chain fragments were detected by SIMS and confirmed the polymer composition. Compared to that of the reference polymer (having only amino acid ester side groups), an increased value of the specific ether carbon groups from PEG confirmed the enrichment of PEG at the surface of PEG-Gal polymers. However, the values were lower than expected. DCA studies showed that the galactose moieties were present at the surface after exposure to an aqueous environment. XPS results confirmed the similarity between experimental and theoretical values for the AH-Gal polymers. This indicated the presence of galactose moieties at the surface, which was confirmed by the DCA data because the contact angles were low compared to those of the other polymers.

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.

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.

Synthesis and characterization of new methacrylate based hydrogels

Hydrogels have been used for several applications, including production of contact lenses, sanitary products and materials for wound dressing. The aim of this work was the development of new methacrylate based hydrogels. These materials present the advantage of being easily produced with different compositions and consequently different permeability and diffusion patterns. Therefore it becomes possible to synthesize hydrogels that can be used to immobilize a variety of compounds, such as drugs, proteins or even cells. During this work new polymers based on methacrylate monomers were prepared. Their characterization was accomplished by several techniques, e.g. Fourier Transform Infrared Spectroscopy (FTIR), swelling measurement, Differential Scanning Calorimetry (DSC) and evaluation of mechanical properties. Their subacute subcutaneous toxicity was also evaluated by using Wistar rats.

Hidrogéis têm sido utilizados para diversas aplicações, incluindo produção de lentes de contato, produtos sanitários e materiais para encerramento de ferimentos. O objetivo deste trabalho consistiu no desenvolvimento de novos hidrogéis a base de metacrilato. Estes materiais apresentam a vantagem de serem facilmente produzidos com diferentes composições e conseqüentemente com distintas permeabilidades e padrões de difusão. Por este motivo, torna-se possível sintetizar hidrogéis que possam ser usados para imobilizar uma grande variedade de compostos, tais como fármacos, proteínas ou mesmo células e tecidos. Neste trabalho foram preparados diferentes polímeros baseados em monômeros de metacrilato. A sua caracterização foi realizada através de diferentes técnicas, como Espectroscopia no Infravermelho com Transformada de Fourier (FTIR), avaliação da capacidade de intumescimento, Calorimetria Diferencial de Varredura (DSC) e avaliação das propriedades mecânicas. A sua toxicidade subaguda subcutânea foi também determinada utilizando ratos Wistar.

Intrauterine levonorgestrel delivered by a frameless system, combined with systemic estrogen: acceptability and endometrial safety after 3 years of use in peri- and postmenopausal women.

OBJECTIVE: To evaluate the acceptability and endometrial safety of a novel intrauterine drug delivery system, FibroPlant-levonorgestrel (LNG), combined with estrogen therapy (ET) in 150 peri- and postmenopausal women, followed-up for at least 3 years. DESIGN: This was a prospective, non-comparative study in peri- and postmenopausal women. In the majority of women, treatment with the FibroPlant-LNG intrauterine system (IUS), combined with ET, was initiated during the perimenopausal transitional phase to establish a smooth transition to menopause and suppress the endometrium to prevent endometrial proliferation and bleeding. A 3.5-cm long coaxial fibrous delivery system, delivering approximately 14 microg LNG/day, was used. The calculated duration of release of the system is at least 3 years. The majority of women received percutaneous 17beta-estradiol (Oestrogel), 1.5 mg daily on a continuous basis, which provides sufficient blood levels of estrogen in most women to suppress climacteric symptoms and protect against bone loss. OUTCOME MEASURES: To measure acceptability, women were asked, after they had the IUS in place for a minimum of 3 years, if they would like to continue the combined regimen and if they would accept renewal of the IUS. Endometrial safety was evaluated by transvaginal ultrasound examination and endometrial biopsy in a subset of 101 women prior to replacement of the IUS. RESULTS: Ninety-four insertions were done in perimenopausal and 56 in postmenopausal women aged between 33 and 78 years. Of the total group of 150 women, 132 women (88.0%) accepted replacement of the IUS and ten are waiting for replacement. This group includes nine women who will receive a second replacement. The number of women continuing the method is 142 (94.6%). Histological examinations conducted on endometrial biopsies from 101 postmenopausal women prior to replacement, after an average period of use of the regimen of 40 months (range 25-50 months), showed predominantly inactive endometrium characterized by pseudodecidual reaction of the endometrial stroma with endometrial atrophia, which is in keeping with the effects seen with a progestogenic compound. There were no specimens showing signs of proliferation. CONCLUSIONS: Results suggest that the frameless FibroPlant-LNG IUS is safe, well tolerated, well accepted and effective in suppressing the endometrium during ET. Intrauterine progestogen administration in postmenopausal women can be regarded as fundamentally advantageous compared with systemically applied progestogens, which may have potentially inherent ill side-effects, especially on the breast and cardiovascular system, as reported in the recent literature.

Encapsulation of osteoblast seeded microcarriers into injectable, photopolymerizable three-dimensional scaffolds based on d,l-lactide and epsilon-caprolactone.

UMR-106 seeded microcarriers were encapsulated into in situ, photopolymerizable three-dimensional scaffolds based on d,l-lactide and epsilon-caprolactone. UMR-106 and rat bone marrow cells proliferated and differentiated well on the microcarriers. The microcarriers were completely colonized after 14 days in culture. The viscous polymer paste allowed to mix the UMR-106 seeded microcarriers and gelatin (porosigen) properly. After the photopolymerization process, microcarriers and gelatin were evenly distributed throughout the scaffold. Gelatin was leached out within 7 h, and a porous scaffold was obtained. The microcarriers remained in the scaffold even after 7 days which demonstrates that they were well entrapped in the polymer. Increasing the amount of entrapped microcarriers (20-50%) leads to scaffolds with a reduced cross-linking. Hence, the microcarriers leached out. The encapsulated UMR-106 cells did not show pyknotic nuclei which demonstrates that the photopolymerization and handling the viscous polymer/gelatin/microcarrier paste is not detrimental for the cells.

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.

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, degradation, and antimicrobial properties of targeted macromolecular prodrugs of norfloxacin.

Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only when it is released in its native form. Also, targeting by using mannose as a homing device is required to achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its native form in vivo, can be transformed into an active drug by targeting.

Development of a miniature, low-dose, frameless intrauterine levonorgestrel-releasing system for contraception and treatment: a review of initial clinical experience.

A low-dose levonorgestrel (LNG)-releasing intrauterine system (IUS) (FibroPlant) has been clinically developed since 1997 for endometrial suppression during hormone replacement therapy in peri- and postmenopausal women, for the treatment of menorrhagia in women with normal uteri or with uterine fibroids, for contraception, for the treatment of endometrial hyperplasia, and for alleviating primary and secondary dysmenorrhoea. Results of preliminary studies confirm the promising nature of this all-round drug delivery system. The low dose of LNG released accounts for the low hormonal side-effect rate and virtual absence of amenorrhoea in premenopausal women. The system has not yet been evaluated in tamoxifen users (to protect the endometrium), or in women with rectovaginal endometriosis. However, early indications suggest that the system will also be suitable for these indications. The frameless drug delivery support of this LNG-releasing IUS has been optimized to reduce the size of the foreign body and to maximize tolerance and continuation of use while simultaneously providing for the maximum duration of action.