Ex vivo porcine vaginal mucosal model of infection for determining effectiveness and toxicity of antiseptics.

AIMS: To develop a semi-high-throughput ex vivo mucosal model for determining efficacy and toxicity of antiseptics. METHODS AND RESULTS: Explants (5 mm) from freshly excised, porcine vaginal mucosa were infected with methicillin-sensitive Staphylococcus aureus (1 × 10(6)  CFU) at the epithelial surface for 2 h. Haematoxylin and eosin staining revealed healthy uninfected tissue and only minor disruptions in tissue infected with methicillin susceptible Staph. aureus (MSSA), which remained in outer epithelial cell layers. After 2 h infection, 10 µl of chlorhexidine digluconate (CHG, 3%), povidone-iodine (PI, 7·5%), octenidine dihydrochloride (OCT, 0·1%) or polyhexamethylene biguanide (PHMB, 0·1%) was applied. Antiseptics significantly reduced MSSA (1-4 log10  CFU/explants) after 0·25 h to 4 h. CHG, PHMB and OCT exhibited persistence at 24 h. In broth culture, CHG 0·012% and PI 0·625% achieved >6 log10 reductions at 2 h. PI-based formulations were more efficacious than unformulated PI. PI-based formulations exhibited no significant cytotoxicity on explants using an MTT assay. CONCLUSIONS: All antiseptics tested in the mucosal MSSA infection model reduced MSSA. CHG and PI were more potent in broth culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a semi-high-throughput mucosal model that can identify compounds or formulations with promising antimicrobial and limited cytotoxic properties.

Interaction of macrophages with fibrous materials in vitro.

Fibrous materials have the potential of being used for tissue scaffolding. The interaction of macrophages with fibres of various compositions and sizes was observed in vitro. The following materials were tested: individual gold fibres; woven fibres of polyester and nylon; non-woven fibres of polybutylene/polypropylene 80:20 and polyester. All fibres had diameters between 2 and 40 microns. At the end of the 24 h incubation time, culture media were retrieved for the assay of tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), two cytokines secreted by activated macrophages. Fibre samples were processed for scanning electron microscopy (SEM), or for immunofluorescence labelling of the MAC-1 and ICAM-1 cell surface markers. Confocal microscopy was used for the latter, which was performed with the woven and non-woven samples. None of the fibre samples induced significant amounts of TNF-alpha or IL-6 secretion in the culture medium, suggesting that the cells did not activate this pathway. SEM on individual gold fibres showed that the fibre diameter had an effect on the morphology of the cells, namely on their extent of spreading. Larger fibres had a higher number of cells, which tended to cluster together without spreading extensively. When comparing woven and non-woven fibres, SEM showed that cells spread extensively on the woven fibres, whereas they tended to maintain their spherical shape on the non-woven fibres. Confocal microscopy demonstrated a difference between materials in the number of MAC-1 and ICAM-1 positive cells. These results demonstrate that a combination of morphological, immune and biochemical markers can be used to distinguish the response of elicited macrophages to various materials. The cells appeared to be only moderately activated on all materials tested, with changes in their morphology but without increased secretion of cytokines. The measured responses imply interactions between nominal fibre composition and fibre diameter.

Expression of intercellular adhesion molecule-1 on macrophages in vitro as a marker of activation.

Macrophage activation is a major component of wound healing. It also determines the extent of inflammatory reactions and the response of the body to implanted materials. We have previously shown, using an in vitro model, that the extent of spreading of macrophages on different materials is a marker of activation, and that a soluble inducer has a dose-response effect on the secretion of cytokines in the culture medium. This work investigates the expression of three different cell surface markers [macrophages MAC-1, MAC-3 and intercellular adhesion molecule-1 (ICAM-1)] on macrophages in vitro using confocal microscopy and shows that ICAM-1 is also a marker of macrophage activation in this model. We observed increased amounts of ICAM-1 on activated macrophages compared to unactivated macrophages, whereas MAC-1 and MAC-3 were either expressed constitutively or demonstrated no quantitative change in expression after activation under the same experimental conditions. We also tested the expression of ICAM-1 with various concentrations of soluble inducers (lipopolysaccharide, 0.001, 0.01, 0.1, 1 and 10 micrograms ml-1. S-27609, 0.1, 0.25, 0.5, 1, 2 and 3 micrograms ml-1 and on a sheet of polylactic acid alone or in combination with soluble inducers. All doses of soluble inducers induced the expression of ICAM-1 on cells grown in glass chamber slides. The induction was not dose related but seemed to work rather in an on-off manner. There was no effect of material on ICAM-1 expression on the cell surface when no soluble inducer was added. This was similar to cytokine secretion, which was not induced by our material alone. When either lipopolysaccharide or S-27609 was used in combination with the material, there was an increase in the average measured intensity of ICAM-1. In this in vitro model, ICAM-1 staining as measured by confocal microscopy is a marker for macrophage activation. Our results suggest that the extent of macrophage activation as measured by ICAM-1 and by cytokine secretion is more sensitive to soluble inducers than to the action of the flat sheet of polylactic acid.

Hydranencephaly in vertebral-basilar territory.

A case is presented of almost complete destruction of the cerebellum secondary to a hemorrhagic event in utero. Lesions consistent with hydranencephaly were found in the territories of the vertebral-basilar circulation. Ependymitis and aqueduct occlusion secondary to the intraventricular bleeding resulted in intrauterine hydrocephalus formation.

Central nervous system reactions to ventriculojugular shunts.

The response of the central nervous system (CNS) to ventricular shunts was examined in 19 patients and was found to be similar to the response observed experimentally in animals and in the cases of wound repair in humans. The major finding was the presence of a fibrous capsule surrounding 7 or 12 shunts in place for longer than 2 months. The CNS results differ from those observed in soft tissue responses by the inconstancy of the fibroblastic reaction. The formation of the envelope, when present, is related to hemorrhage secondary to insertion, with no relationship to the duration of shunt use. The ability of tumor cells to spread along the shunt path reflects the weak adhesion between the neural elements and the shunt.

Monocyte interactions with solid substrates monitored by chemiluminescence.

The kinetics of interaction of solid substrates with mononuclear cells is a little explored by important feature in the evaluation of potential prosthetic materials. Monocyte production of chemiluminescence in a luminol-enhanced system was used to explore such kinetics. A theoretical model of cell/surface interaction is developed and alternatives in the evaluation of data generated by chemiluminescent curves are presented. The interactions of monocytes with a polytetrafluoroethylene (PTFE) surface were similar to those seen in studies on phagocytosis and were dependent on an absorbed serum protein layer. This study illustrates the use of a simple laboratory test for monocyte oxidative function in assessing the potential inflammatory effects of various prosthetic materials.

Platelet adhesion to novel phospholipid materials: modified phosphatidylcholine covalently immobilized to silica, polypropylene, and PTFE materials.

Based on the premise of achieving blood compatibility through mimicking the chemical constitutents of the biologically insert surface of the unactivated platelet membrane, a process was developed that entails the covalent grafting of modified phosphatidylcholine molecules to materials including silica, polypropylene, and polytetrafluoroethylene (PTFE) polymer films. These materials were characterized using x-ray photoelectron spectroscopy (XPS) and contactangle measurements. The phosphatidylcholine-containing materials (PC materials) were used as substrates in the plateletadhesion assays and were subjected to enzymatic degradation evaluation. Phosphatidylcholine-grafted silica materials do not support platelet adhesion. In addition the number of adherent platelets correlate with the amount of grafted phospholipid present, as indicated by the phosphorus/ carbon ratio obtained by XPS analysis. Platelet adhesion to phosphatidylcholine-grafted polypropylene and PTFE was inhibited 80% and 90%, respectively, when compared with platelet adhesion to unmodified polypropylene and PTFE.

Histological characterization of a delayed wound healing model in pig.

Chronic wounds, such as venous ulcers and pressure ulcers, frequently remain unresponsive to currently available treatments. Several animal models of wound healing have been published, including models of impaired healing developed to mimic the clinical condition of chronic wounds better. We used a delayed wound healing model in the pig that uses irradiation of the skin prior to creation of the surgical wounds and characterized it histologically. Radiation was used on one side of the back prior to making four full-thickness wounds on each side. Clinical observations were performed to record granulation tissue, reepithelialization, and wound area as a function of time. Histology data were obtained at 1, 2, 3, and 4 weeks, and slides were stained with hematoxylin and eosin for general observations. Immunohistochemistry was performed using laminin as a marker for blood vessels, and the number, size, and circularity of blood vessels found in the granulation tissue were measured. Our results show that this model causes a delay in wound healing that is mostly apparent between days 7 and 15. Granulation tissue took more time to form and fill the wounds on the irradiated side, and blood vessels were slower to develop. Blood vessels were larger and more irregular in shape on the irradiated side than on the control side. After 2 weeks, healing resumed, indicating that the induced damage was not irreversible. These results suggest that this model can be used to test the effects of therapeutic approaches intended to treat chronic wounds.