Ex vivo and in vivo evaluation of residual chlorhexidine gluconate on skin following repetitive exposure to saline and wiping with 2% chlorhexidine gluconate/70% isopropyl alcohol pre-operative skin preparations.

BACKGROUND: Skin antisepsis is performed before surgery to minimize the risk of surgical site infections. Chlorhexidine gluconate (CHG) is routinely used in this application, but it may be removed during surgery when prepped areas are exposed to fluid and repeated blotting. AIM: This work evaluated the effect of adding a film-forming acrylate copolymer to a CHG-containing skin preparation on minimizing CHG loss during a simulated surgical irrigation and wiping procedure. The results were compared with those obtained with a commercially available water-soluble CHG preparation. METHODS: Two studies using excised porcine skin and one study on human volunteers were performed. In each study, the CHG preparations were applied and the treated sites were challenged with repetitive saline soaks and gauze dabbing to simulate surgical conditions. Challenged and unchallenged sites were analysed either for CHG content by high-performance liquid chromatography, or for bacterial log recovery after seeding an indicator organism (reflecting remaining CHG activity). FINDINGS: After irrigation and wiping, skin treated with the film-forming CHG preparation had more CHG remaining both on excised pig skin and in the human model. In the pig model, there was a lower recovery of inoculated bacteria with the CHG preparation containing the film-forming copolymer. No skin irritation or adverse events were reported in the human study. CONCLUSIONS: The addition of a film-forming copolymer has the potential to improve the retention of CHG on skin throughout a surgical procedure compared to a water-soluble preparation. This improved retention may lead to better antimicrobial activity.

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.

Subconjunctival biocompatibility of a viscous bioerodable poly(ortho ester).

The biocompatibility of a viscous poly(ortho ester) (POE) intended for prolonged intraocular drug delivery was studied. This hydrophobic and bioerodable carrier was subconjunctivally injected in rabbits and evaluated both clinically and histologically. To assess the cause of the triggered transient acute inflammatory reaction, the two monomers, the intermediate and final degradation products, and the local toxicity of different solvents used during the polymer preparation were tested. Since the two initial monomers and the intermediate degradation products induced only moderate inflammation, the main acute inflammatory reaction is attributed to the formation of an acidic by-product which has been monitored in vitro by measuring the progressive decrease of the environmental pH. The influence of the sterilization procedure on tissue biocompatibility was established by comparing two polymers of similar molecular weight: one after gamma-sterilization, and an aseptically synthesized one. The biocompatibility was significantly improved by avoiding irradiation of the polymer.

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.

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.

Biocompatibility of a new semisolid bioerodible poly(ortho ester) intended for the ocular delivery of 5-fluorouracil.

The biocompatibility of a new semisolid, hydrophobic poly(ortho ester) (POE) intended for controlled drug delivery to the eye was evaluated. The polymer was injected subconjunctivally in rabbits, and clinical and histologic examinations were performed 3, 10, 15, and 21 days after injection. Polymers injected as controls were an aqueous gel of sodium hyaluronate (SH), 1% in phosphate buffer, and medical grade silicone oil. After injection, the POE emulsified into small droplets and a focal eosinophilic reaction was noted at 3 days' implantation. At 10 days' implantation, the POE was not identified in the implantation site and the inflammatory reaction had resolved, with fibroblasts being the predominant cell type. At 15 and 21 days, no POE was identified and normal appearing tissue was present in the injection site. Sodium hyaluronate was not inflammatory over the period of the implantations. Silicone oil induced a slight inflammation at 3 days, with the presence of eosinophils and limited necrosis with cellular debris. Silicone oil was present in the implantation site at 3, 10, 15, and 21 days. The inflammatory response to the respective polymers was evaluated in the subconjunctival tissue. The inflammatory reaction was quantified at the implant site, adjacent subconjunctival tissues, and scleral and corneal stroma. The inflammatory cell densities in these respective tissue zones were determined, and the ratio of eosinophils over total inflammatory cells was calculated. POE did not become encapsulated with fibrous tissue, but biodegraded in a short time, indicating its potential for use after glaucoma filtration surgery.

Effects of EGF, IL-1 and their combination on in vitro corneal epithelial wound closure and cell chemotaxis.

We investigated the effects of EGF, IL-1 and their combination on closure of wounds inflicted on rabbit corneal epithelial cell cultures and on migration of these cells in microchemotaxis chambers. In vitro corneal epithelial wound closure depended on the applied concentrations of EGF or IL-1. Twenty-four hours after wounding, the smallest wounds were obtained with 50 ng ml-1 of EGF and 1 ng ml-1 of IL-1, respectively. The effect on wound closure of combinations of EGF and IL-1 was additive even at concentrations that were optimal for each growth factor when applied alone. We found that EGF increases the chemotactic migration of rabbit corneal epithelial cells. Cell chemotaxis depended both on the concentration of EGF and on the number of cells applied in the assay. This response to EGF was seen at concentrations that were effective in the wound closure assay. The magnitude of the chemotactic migration response was much smaller with IL-1 than with EGF. Similarly to the observations on wound closure, the effect on cell chemotaxis of combinations of EGF and IL-1 was additive. The ability of EGF, and EGF/IL-1 combinations to modulate corneal epithelial cell chemotactic migration supports migration as a possible biological mechanism of the acceleration of corneal epithelium wound closure by these drugs.

Biotolerance of a semisolid hydrophobic biodegradable poly(ortho ester) for controlled drug delivery.

We evaluated the biotolerance of a new semisolid poly(ortho ester) (POE) intended for controlled drug delivery. Two different investigations were carried out in rats: subcutaneous injections and the cage-implant system. Sodium hyaluronate (SH), 1%, in phosphate buffer was used as a noninflammatory control. When injected subcutaneously in rats, the POE induced a mild and local inflammation at 3 and 7 days followed by a minimal chronic inflammation at 14 and 21 days. When using the cage-implant system, we quantified the inflammatory components of the exudate surrounding the implanted material within the cage system at 3, 7, 14, and 21 days. The total leukocyte and macrophage concentrations were higher only at 7 days for both SH and POE compared to the control (empty cage). The other parameters were not significantly different from the control. These results show that POE is well-tolerated by rats when used subcutaneously.

Sodium hyaluronate 0.25% used as a vehicle increases the bioavailability of topically administered gentamicin.

Sodium hyaluronate (SH) solutions have a longer precorneal residence time than isotonic saline solution in rabbits and in humans. The present study investigates the effect of a 0.25% SH vehicle, compared with a phosphate buffer solution (PBS), on the tear concentration after topical administration of gentamicin sulfate (GS) in humans. Eight volunteers received 25 microliters 0.5% GS in PBS in the left eye and 25 microliters 0.5% GS in 0.25% SH in the right eye. Tear samples of 1 microliters were taken from the inferior sulcus using a capillary before instillation and 5, 10, 20, and 40 min after instillation. The tear concentration of GS was determined by radioimmunoassay. There was a statistically significantly higher concentration of GS in the inferior conjunctival sulcus of the SH-instilled eye at 5 min (P < 0.01) and at 10 min (P < 0.05) after instillation (paired t-test, n = 9). At 20 min the concentration of GS in the SH-instilled eye was only slightly higher, and at 40 min GS concentrations in left and right eyes were comparable. SH 0.25% used as a vehicle therefore increases the availability of GS at the ocular surface for at least 10 min. This effect could be due to the prolonged precorneal residence time of SH.

Urokinase-type plasminogen activator in human aqueous humor.

In the anterior segment of the eye, fibrin clots must be rapidly resorbed to prevent further fibrosis and scarring. The aqueous humor of patients undergoing cataract surgery was analyzed for the presence of components of the fibrinolytic cascade. In 30 patients, aqueous humor and plasma were compared for their content of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activators inhibitors (PAIs), plasminogen, and total proteins. With gel electrophoresis and zymographic assays of serial dilutions of plasma and aqueous humor, all these components were found to be present at lower concentrations in aqueous humor than in plasma. For total proteins, the aqueous/plasma ratio was approximately 0.003, and for plasminogen it was 0.001. Interestingly, the aqueous/plasma ratio for uPA was not as low and varied from 0.01 to 0.03. A significant proportion of the uPA in aqueous humor was present in the two-chain active form. In addition to uPA, aqueous humor contained lower levels of tPA, but no detectable levels of reactive plasminogen activators inhibitors (PAIs). The presence of a relatively high concentration of active uPA shows that the proteolytic balance of the aqueous humor in the anterior chamber of the eye is shifted toward fibrinolysis.