Theoretical and Experimental Elucidation of the Adsorption Process of a Bioinspired Peptide on Mineral Surfaces.

Inorganic materials used for biomedical applications such as implants generally induce the adsorption of proteins on their surface. To control this phenomenon, the bioinspired peptidomimetic polymer 1 (PMP1), which aims to reproduce the adhesion of mussel foot proteins, is commonly used to graft specific proteins on various surfaces and to regulate the interfacial mechanism. To date and despite its wide application, the elucidation at the atomic scale of the PMP1 mechanism of adsorption on surfaces is still unknown. The purpose of the present work was thus to unravel this process through experimental and computational investigations of adsorption of PMP1 on gold, TiO2, and SiO2 surfaces. A common mechanism of adsorption is identified for the adsorption of PMP1 which emphasizes the role of electrostatics to approach the peptide onto the surface followed by a full adhesion process where the entropic desolvation step plays a key role. Besides, according to the fact that mussel naturally controls the oxidation states of its proteins, further investigations were performed for two distinct redox states of PMP1, and we conclude that even if both states are able to allow interaction of PMP1 with the surfaces, the oxidation of PMP1 leads to a stronger interaction.

Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair.

The recent advances in 3D-printed silicone (PDMS: polydimethylsiloxane) implants present prospects for personalized implants with highly accurate anatomical conformity. However, a potential adverse effect, such as granuloma formation due to immune reactions, still exists. One potential way to overcome this problem is to control the implant/host interface using immunomodulatory coatings. In this study, a new cytokine cocktail composed of interleukin-10 and prostaglandin-E2 was designed to decrease adverse immune reactions and promote tissue integration by fixing macrophages into M2 pro-healing phenotype for an extended period of time. In vitro, the cytokine cocktail maintained low levels of pro-inflammatory cytokine (TNF-α and IL-6) secretions and induced the secretion of IL-10 and the upregulation of multifunctional scavenging and sorting receptor stabilin-1, expressed by M2 macrophages. This cocktail was then loaded in a gelatine-based hydrogel to develop an immunomodulatory material that could be used as a coating for medical devices. The efficacy of this coating was demonstrated in an in vivo rat model during the reconstruction of a tracheal defect by 3D-printed silicone implants. The coating was stable on the silicone implants for over 2 weeks, and the controlled release of the cocktail components was achieved for at least 14 days. In vivo, only 33% of the animals with bare silicone implants survived, whereas 100% of the animals survived with the implant equipped with the immunomodulatory hydrogel. The presence of the hydrogel and the cytokine cocktail diminished the thickness of the inflammatory tissue, the intensity of both acute and chronic inflammation, the overall fibroblastic reaction, the presence of oedema and the formation of fibrinoid (assessed by histology) and led to a 100% survival rate. At the systemic level, the presence of immunomodulatory hydrogels significantly decreased pro-inflammatory cytokines such as TNF-α, IFN-γ, CXCL1 and MCP-1 levels at day 7 and significantly decreased IL-1α, IL-1ß, CXCL1 and MCP-1 levels at day 21. The ability of this new immunomodulatory hydrogel to control the level of inflammation once applied to a 3D-printed silicone implant has been demonstrated. Such thin coatings can be applied to any implants or scaffolds used in tissue engineering to diminish the initial immune response, improve the integration and functionality of these materials and decrease potential complications related to their presence.

Step-by-step build-up of covalent poly(ethylene oxide) nanogel films.

Hydrogels based on poly(ethylene glycol) (PEG) are commonly used for studies related to cell fate and tissue engineering. Here we present a new covalent layer-by-layer build-up process leading to PEG coatings of nanometer size called "nanogel films". Compared to macroscopic hydrogels, such nanogels should provide a fine control over the structure and the thickness of the coating. Alternated deposition of bifunctional and tetra functional PEG molecules reacting through thiol/maleimide click chemistry is evaluated by quartz crystal microbalance. We first study parameters influencing the build-up process of such coatings and demonstrate the importance of (i) the nature of the first deposited layer, (ii) the PEG concentrations and (iii) the length of the PEG chains that appears to be the most significant parameter influencing film growth. The build-up process can be extended to a large variety of substrates like SiO2 or polymers by using an appropriate anchoring layer. Covalent functionalization of these nanogel films by proteins or enzymes is suited by modifying the biomolecules with thiol or maleimide groups and immobilizing them during the build-up process. Activity of the embedded enzymes can be maintained. Moreover ligands like biotin can be incorporated into the film and recognition by streptavidin can be modulated by playing with the number of PEG layers covering biotin. Compared to well-known PEG hydrogels, these new coatings are promising as they allow to (i) build thin nanometric coatings, (ii) finely control the amount of deposited PEG and (iii) organize the position of the embedded biomolecules inside the film layers.

Double entrapment of growth factors by nanoparticles loaded into polyelectrolyte multilayer films.

Delivery of growth factors and control of vascularization are prominent problems in regenerative medicine. Vascular endothelial growth factor (VEGF) has been used both in vitro and in vivo to promote angiogenesis but due to its short half-life its controlled delivery is a sought after method. In this study we present a new concept of degradable drug loaded nanoparticles entrapped into exponentially growing multilayer films. Through hydrolysis of the nanoparticles, the drug can be delivered over long periods in a controlled manner. Poly(ε-caprolactone) nanoparticles were loaded with VEGF and in turn the release of VEGF from a surface is controlled by a thick layer-by-layer polyelectrolyte film. Direct loading of VEGF inside the film was not efficient for long-term applications. When VEGF loaded nanoparticles were introduced into the film, the particles were equally distributed inside and were stable after several washes. Moreover, the presence of the film sustained the release of VEGF for 7 days. Addition of the nanoparticles to the film promoted endothelial cell proliferation, mainly due to the presence of VEGF. Mechanical properties of the film (Young's moduli) were also improved by the presence of nanoparticles. However, in the presence of the film loaded with nanoparticles and without any direct contact with this film, endothelial cell growth was also enhanced on polystyrene and on Transwell insert surfaces which demonstrates the effectiveness of the nanoparticles not only to improve the mechanical properties of the film but also to deliver active VEGF. An increase in nitric oxide levels as an indicator of endothelial cell activity was monitored and was correlated with the release of VEGF from the nanoparticle/film platform. Finally, such a system can be used as an auxiliary delivery body within implants to finely control the release of bioactive agent containing nanoparticles.

Rat nerve regeneration with the use of a polymeric membrane loaded with NGF.

Exogenous neurotrophic factors, delivered by various systems, are used to improve nerve regeneration. This study tested the effectiveness of a polymeric membrane loaded with Nerve Growth Factor (NGF) on mental nerve regeneration after a crush injury in rats. We tested NGF application, known to play a role in afferent fiber repair in dental neurobiology, to see if it could improve the regeneration. Afferent neurogram recordings and histological analyses of the trigeminal ganglion neurons were performed. One month after the crush injury, early regeneration was observed independently of exogenous NGF. However, as compared with the activity level recorded before the injury, the afferent activity was reduced by 28.5% without NGF, and the mean number of labeled neurons decreased. With NGF, activity was increased by 30.8%, with no significant histological difference compared with animals without lesions. NGF application through a polymeric membrane can influence degenerative and/or regenerative processes after a crush injury.

Development of tracheal prostheses made of porous titanium: a study on sheep.

Authors report the development of a biomaterial to be used for tracheal and laryngeal reconstruction. This experimentation follows the replacement of trachea in rats with porous titanium implants. The aim of the study is to test this type of prosthesis on sheep, whose trachea is of comparable size to that of humans. Six ewes were implanted with porous titanium implants after resection of 5 cm of trachea. The planned period for the implantation was from 3 to 6 months before the sacrifice of the animals for histological analysis. After a simple immediate postoperative course, the implantations developed complications of tracheal patency, responsible for four deaths (tracheal obstruction by mucous plug n = 2, inferior necrosis of trachea n = 1, pneumopathy n = 1). The two remaining sheep presented no complications. The mechanical performance of the prostheses was good. The histological results showed an inflammatory stenosis of the tracheo-prosthetic junctions, which was not the direct result of death. The protheses were integrated by the surrounding tissue, but endoprosthetic colonisation by pseudostratified ciliated columnar epithelium was low or nil. The absence of endoprosthetic lining was responsible for the complications. The biocompatibility of the biomaterial is not in question, but the surgical procedure will have to be modified by an endoprosthetic mucous graft before implantation so as to accelerate healing process.

In vitro biocompatibility of different polyester membranes.

Nowadays, synthetic biodegradable polymers, such as aliphatic polyesters, are largely used in tissue engineering. They provide several advantages compared to natural materials which use is limited by immunocompatibility, graft availability, etc. In this work, poly(L-lactic) acid (PLLA), poly(DL-lactic) acid (PDLA), poly-epsilon-caprolactone (PCL), poly(L-lactic)-co-caprolactone (molar ratio 70/30) (PLCL) were selected because of their common use in tissue engineering. The membranes were elaborated by solvent casting. Membrane morphology was investigated by atomic force microscopy. The membranes were seeded with human fibroblasts from cell line CRL 2703 in order to evaluate the biocompatibility by the Alamar blue test. The roughness of the membranes ranged from 4 nm for PDLA to 120 nm and they presented very smooth surface except for PCL which beside a macroscopic structure due to its hydrophobicity. Human fibroblasts proliferated over 28 days on the membranes proving the non-in vitro toxicity of the materials and of the processing method. A further step will be the fabrication of three-dimensional scaffold for tissue engineering and the treatment of the scaffolds to augment cell adhesion.

Multiple and time-scheduled in situ DNA delivery mediated by beta-cyclodextrin embedded in a polyelectrolyte multilayer.

The basic premise of gene therapy is that genes can be used to produce in situ therapeutic proteins. The controlled delivery of DNA complexes from biomaterials offers the potential to enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment. Immobilization of the DNA to the substrate to which cells adhere maintains the DNA in the cell microenvironment for subsequent cellular internalization. Here, layer-by-layer (LBL) films made from poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) containing DNA were built in the presence of charged cyclodextrins. The biological activities of these polyelectrolyte films were tested by means of induced production of a specific protein in the nucleus or in the cytoplasm by cells in contact with the films. This type of coating offers the possibility for either simultaneous or sequential interfacial delivery of different DNA molecules aimed at cell transfection. These results open the route to numerous potential applications in patch vaccination, for example.

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers.

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an "in" and "out" diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

[Study of 50 patients with sporotrichosis. Clinical and laboratory assessment]. / Estudio de 50 pacientes con esporotricosis. Evaluación clínica y de laboratorio.

Because sporotrichosis is the most frequent subcutaneous mycosis in Mexico and the clinical aspect is not always characteristic, the aim of this study was to evaluate laboratory diagnosis techniques. Fifty patients with clinical diagnosis of subcutaneous sporotrichosis were studied including clinical and epidemiologic data. Metabolic antigen was used to elicit delayed hypersensitivity skin reaction in all patients. Exudate was plated on Sabouraud agar and biopsy material was submitted to indirect immunofluorescence and histopathology. Results showed that sporotrichosis frequency was higher in women (62%), in children and adolescents under 20 years of age (34%) and adults older than 50 years of age (28%). Disease was predominant in farmers (44%) followed by housewives (30%). Lymphangitic form accounted for 82% of cases and these were localized in upper limbs (54%). In 66% of cases, histopathology showed S. schenckii yeasts; hypersensitivity skin reaction was positive in 76% and culture in 94%. By indirect immunofluorescence, parasitic elements were demonstrated in all patients corresponding to both sensitivity and specificity 100%. In this work, indirect immunofluorescence was the most efficient sporotrichosis diagnostic method followed by culture, hypersensitivity skin reaction, and histopathologic study.

Extended random sequential adsorption model of irreversible deposition processes: from simulations to experiments.

An experimental study of the irreversible deposition of colloidal particles of various radii R on a solid surface is presented over a wide range of the Péclet number, Pe, or reduced radius R* (Pe = R*(4)). The experimental data are analyzed by means of a new generalized random sequential adsorption model that takes explicitly the diffusion of the particles during the deposition into account. It allows description of the continuous transition from a random sequential adsorption-like to a ballistic-like deposition behavior. It depends on three parameters: d(s), related to the diffusion of the particles before adhesion; n(s), related to the number of allowed adhesion trials of a particle; and R(e), representing the effective particle radius. The model allows accounting for all of the experimental observations relative to the radial distribution functions and the number density fluctuations over the whole coverage range and all investigated values of R*. In addition, it is found that d(s)/R is proportional to R*(-2) as expected for a diffusional process. Moreover, the parameters d(s) and n(s) appear to be connected through the empirical relation (d(s)/R)n(s)(2/3) = C, where C is found to be of the order of 50. This unique statistical model allows an accurate description of the irreversible deposition process, whatever the influence of gravity with respect to diffusion.

Adhesion Probability of Colloidal Particles with Repulsive Soft Interaction.

Computer simulations of the irreversible adhesion of charged colloidal particles at a solid/liquid interface are performed to determine whether the distribution of particles in the vicinity of a preadsorbed (also charged) one follows the Boltzmann law applied to an a priori uniform adhesion probability, as first assumed by Adamczyk et al. (J. Colloid Interface Sci. 140, 123 (1990)). If true, this would indicate that the whole information on the deposition process is contained in the potential energy distribution on the adsorbing surface. In general, diffusion in a field of force and the irreversibility of the process induce significant deviations from the Boltzmann-weighted uniform adhesion density. Nevertheless, it is shown that for particles characterized by a small gravitational energy this procedure leads to a reasonable first approximation of the distribution of the particles over the adsorbing surface. This observation thus demonstrates the validity of Adamczyk's assumption and extends its range of applicability to the case of a weak gravitational field. Copyright 1999 Academic Press.

Red blood cell adhesion on a solid/liquid interface: comparison of two models.

Discoid red blood cells (RBCs) deposited irreversibly on a horizontal glass surface are studied by means of optical microscopy and image analysis. The relative surface covered by the RBCs, as well as the variance of this surface coverage as a function of the cell concentration, are analyzed and compared to the results derived from the ballistic deposition (BD) model. This model describes the irreversible deposition of spherical particles under the influence of an infinitely large gravitation force and does not allow for overlaps between adsorbed particles. In spite of these characteristics, the BD model permits, surprisingly, to reproduce our experimental observations on the deposition of RBCs on a flat surface. This finding is discussed, in particular in respect to a former study where a model was developed for colloidal particles of this particular geometric shape.

Red blood cell adhesion on a solid/liquid interface.

Red blood cells (RBCs), previously fixed with glutaraldehyde, adhere to glass slides coated with fibrinogen. The RBC deposition process on the horizontal glass surface is investigated by analyzing the relative surface covered by the RBCs, as well as the variance of this surface coverage, as a function of the concentration of particles. This study is performed by optical microscopy and image analysis. A model, derived from the classical random sequential adsorption model, has been developed to account for the experimental results. This model highlights the strong influence of the hydrodynamic interactions during the deposition process.

[Epidemiology of mycetoma in Mexico: study of 2105 cases]. / Epidemiología del micetoma en México: estudio de 2105 casos.

A survey was carried out in Mexico to determine the incidence and epidemiological characteristics of mycetoma. Data was collected from a total of 2105 cases of mycetoma throughout a 30 year period (1956-1985), with an average incidence of 70 cases per year. Results showed a sex distribution of 76.1% male and 23.9% females. Age distribution indicated a 35% between 16 to 30 and 23% between 31 to 40 year old population. Most cases occurred in land-workers (60.2%) and in housewives with rural residence (21.3%). Lesions occurred most frequently in lower limbs (64.1%), trunk (17.4%) and upper limbs (13.6%). The geographic distribution within Mexico revealed that the States with the highest incidence were: Jalisco, Nuevo León, San Luis Potosi, Morelos and Guerrero The predominant etiologic agents found 97.8% corresponded to actinomycetes, from which Nocardia brasiliensis (86.6%) and Actinomadura madurae (10.2%) showed the higher frequency. Eumycetoma (2.2%) was due to Madurella grisea and M. mycetomatis in most cases.

[Epidemiologic study of mycetomas in Mexico. Apropos of 502 cases]. / Etude épidémiologique des mycétomes au Mexique. A propos de 502 cas.

The epidemiological data of 502 mycetomas studied in the Department of Mycology, "Centro Dermatológico Pascua", Mexico City, were analysed. Mycetomas prevail in males (79.7%), they are more frequent between 16 and 45 years of age (75%) and among rural workers (62.5%); they preferentially affect lower limbs (62.5%); these data are generally similar to the known publications on the matter. Actinomycetomas are the most frequent with 97.2% of the cases, distributed as follows: Nocardia: 85.6% among which 71.9% are N. brasiliensis, Actinomadura madurae: 9.6%, Streptomyces somaliensis: 1.6%, Actinomadura pelletieri: only one case: 0.2%. Eumycetomas, a total of 9, are due to Madurella mycetomi (2), Madurella grisea (2), an undetermined black grain (1), Acremonium sp. and Fusarium sp. (1), Fusarium, sp. (1), Pseudallescheria boydii (1), and an undetermined white grain (1).

Itraconazole for deep mycoses: preliminary experience in Mexico.

Itraconazole has been used as oral therapy for deep mycoses since July 1984 in our institution. So far, therapy has been evaluated for eight patients: one with cutaneous coccidioidomycosis; two with chromomycosis (Fonsecaea pedrosoi); and five with sporotrichosis (three fixed, one lymphangitic, and one disseminated). Clinical and mycologic evaluations were done at the beginning of treatment, 15 days after the initiation of treatment, and monthly thereafter. Dosage was 100-200 mg per day, and duration of treatment was based on response. The cases of coccidioidomycosis and lymphangitic sporotrichosis were mycologically cured after two and seven months of treatment with 100 mg per day, respectively. The six other patients required higher doses. Two patients with sporotrichosis and one with chromomycosis were cured, and one patient with sporotrichosis and one with chromomycosis were markedly improved. In one patient with sporotrichosis, treatment was discontinued because of the slow response. No adverse reactions to treatment were reported.

Tratamiento de las onicomicosis con miconazol barniz. / Treatment of onychomycosis with miconazol varnish

Se hace un estudio de 30 pacientes con onicomicosis tratados con miconazol barniz en solucion alcoholica al 2%. Solo se incluyeron pacientes con examen directo y cultivo positivo. El 40% correspondio a T. unguium y 60% a candidiasis ungueal. La evolucion vario de 5 meses a 5 anos. En 5 pacientes estaban afectadas las unas de manos, en 12 las de pies y en 13 ambas. Se observaron resultados excelentes en 23.3%, buenos en 56.7% y malos en 20%. El tiempo minimo de curacion completa fue de 4 meses y el maximo de observacion de 6 meses. La mejoria se observo mas rapidamente en los casos por C. albicans. El numero total de casos con mejoria clinica y negativizacion micologica fue de 80%. Se subraya la ventaja de este medicamento por su uso topico y por su accion polivalente sobre dermatofitos y C. albicans y porque acorta el tiempo de tratamiento de las onicomicosis, en comparacion con otros medicamentos en uso