A collagen-based layered chronic wound biofilm model for testing antimicrobial wound products.

A new in vitro chronic wound biofilm model was recently published, which provided a layered scaffold simulating mammalian tissue composition on which topical wound care products could be tested. In this paper, we updated the model even further to mimic the dynamic influx of nutrients from below as is the case in a chronic wound. The modified in vitro model was created using collagen instead of agar as the main matrix component and contained both Staphylococcus aureus and Pseudomonas aeruginosa. The model was cast in transwell inserts and then placed in wound simulating media, which allowed for an exchange of nutrients and waste products across a filter. Three potential wound care products and chlorhexidine digluconate 2% solution as a positive control were used to evaluate the model. The tested products were composed of hydrogels made from completely biodegradable starch microspheres carrying different active compounds. The compounds were applied topically and left for 2-4 days. Profiles of oxygen concentration and pH were measured to assess the effect of treatments on bacterial activity. Confocal microscope images were obtained of the models to visualise the existence of microcolonies. Results showed that the modified in vitro model maintained a stable number of the two bacterial species over 6 days. In untreated models, steep oxygen gradients developed and pH increased to >8.0. Hydrogels containing active compounds alleviated the high oxygen consumption and decreased pH drastically. Moreover, all three hydrogels reduced the colony forming units significantly and to a larger extent than the chlorhexidine control treatment. Overall, the modified model expressed several characteristics similar to in vivo chronic wounds.

Current In Vitro Biofilm-Infected Chronic Wound Models for Developing New Treatment Possibilities.

Significance: The prevalence of chronic wounds is increasing worldwide. The most recent estimates suggest that up to 2% of the population in the industrialized countries is affected. Recent Advances: During the past few decades, bacterial biofilms have been elucidated as one of the primary reasons why chronic wounds fail to heal. Critical Issues: There is a lack of direct causation and evidence of the role that biofilms play in persistent wounds, which complicates research on new treatment options, since it is still unknown which factors dominate. For this reason, several different in vitro wound models that mimic the biofilm infections observed in chronic wounds and other chronic infections have been created. These different models are, among other purposes, used to test a variety of wound care products. However, chronic wounds are highly complex, and several different factors must be taken into consideration along with the infection, including physiochemical and human-supplemented factors. Furthermore, the limitations of using in vitro models, such as the lack of a responsive immune system should always be given due consideration. Future Directions: Present understandings of all the elements and interactions that take place within chronic wounds are incomplete. As our insight of in vivo chronic wounds continues to expand, so too must the in vitro models used to mimic these infections evolve and adapt to new knowledge.

Measuring enzymatic degradation of degradable starch microspheres using confocal laser scanning microscopy.

Degradable starch microspheres (DSM) have long been used for topical haemostasis, temporary vascular occlusion and as drug delivery systems. When used for the latter, exact degradation rates of DSM have high importance, as this ensures a controlled and timed drug delivery. Current methods of analysing degradation rates are based on whole batch measurements, which does not yield information regarding individual times of degradation nor does it provide direct correlation measurements between sphere diameter and specific degradation time. In this paper we present an alternative method for measuring degradation rates of biodegradable starch microspheres using confocal laser scanning microscopy (CLSM). We succeeded in visualizing the degradation by staining the DSM and then following the spheres over time in a confocal microscope, after the addition of α-amylase. Individual degradation rates of single spheres could be followed, allowing a precise correlation measure between sphere size and degradation time. Furthermore, physical abnormalities such as internal cavities were detected within some spheres. These physical differences also had a measurable effect on the rate of degradation. Finally, complete degradation rates could be determined very accurately. To our knowledge, this is the first paper in which DSM degradation is visualized and measured using CLSM. STATEMENT OF SIGNIFICANCE: Using degradable starch microspheres as a drug delivery system, is a continuously evolving field which shows promise in several different areas of illnesses. This paper presents a new method which visualizes enzymatic degradation of starch microspheres in real-time using confocal microscopy. The method is simple, yet the versatility of it suggests that it could be broadly applied within the field of biodegradation. Here, it illuminates a previously uninvestigated parameter: the effect of physical sphere deformities on the rate of degradation. It also provides precise correlation measures between initial sphere size and time of complete degradation.