Sucrose-mediated formation and adhesion strength of Streptococcus mutans biofilms on titanium.

Biofilms consist of bacterial cells surrounded by a matrix of extracellular polymeric substance (EPS), which protects the colony from many countermeasures, including antibiotic treatments. Growth and formation of bacterial biofilms are affected by nutrients available in the environment. In the oral cavity, the presence of sucrose affects the growth of Streptococcus mutans that produce acids that erode enamel and form dental caries. Biofilm formation on dental implants commonly leads to severe infections and can restrict osseointegration necessary for the implant to be successful. This work determines the effect of sucrose concentration on biofilm EPS formation and adhesion of Streptococcus mutans, a common oral colonizer, to titanium substrates simulating common dental implants. Biofilm formation and profiles are visualized at high magnification with scanning electron microscopy (SEM). Large mounds and complex structures consisting of bacterial cells and EPS can be seen in biofilms at sucrose concentrations that are favorable for biofilm growth. The laser spallation technique is used to apply stress wave loading to the biofilm, causing the biofilm to delaminate at a critical tensile stress threshold. The critical tensile stress threshold is the adhesion strength. Because laser spallation applies the stress loading to the rear of the substrate, bulk adhesion properties of the biofilm can be determined despite the heterogenous composition and low cohesion strength of the biofilm. Statistical analysis reveals that adhesion strength of biofilms initially increase with increasing sucrose concentration and then decrease as sucrose concentration continues to increase. The adhesion strength of bacterial biofilms to the substrate in this study is compared to the adhesion of osteoblast-like cells to the same substrates published previously. When sucrose is present in the biofilm growth environment, S. mutans adhesion is higher than that of the osteoblast-like cells. Results of this study suggest sucrose-mediated S. mutans biofilms may outcompete osteoblasts in terms of adhesion during osseointegration, which could explain higher rates of peri-implant disease associated with high sugar diets. Further studies demonstrating adhesion differentials between biofilms and cells including co-cultures are needed and motivated by the present work.

Exploring microwave irradiation as a method to disinfect contact lens cases.

PURPOSE: Biofilm formation caused by infrequent contact lens case replacement and the ineffectiveness of multi-purpose solutions (MPS) on biofilm removal is associated with high rates of bacterial keratitis infections. This study demonstrated biofilm elimination from the contact lens case by microwave irradiation. METHODS: Staphylococcus aureus biofilms indicative of 3-9 months of contact wear were cultured in contact lens cases and visualized with crystal violet (CV) staining. Biofilms in contact cases were then exposed to four treatment regimens: No treatment (n = 8), 45 s microwave irradiation (n = 8), tap water (n = 6), and MPS (n = 9). Bacterial survival was assessed by colony forming unit (CFU) assay using streak dilutions. RESULTS: Visualization of the biofilms through CV staining revealed that biofilms coalesce between ribs of the contact case. In 5/8 cases no CFU were cultivated from the case after treatment with microwave irradiation. In tap water and MPS the first dilution averaged 6 ± 2 and 31 ± 13 CFUs per plate, respectively, while microwave irradiation averaged < 1 CFU per plate. In Dilution 2, the average reduced to 0.7 ± 0.7 and 6 ± 5 CFUs per plate for tap water and MPS, respectively, while microwave irradiation had 0 CFUs in Dilution 2. CONCLUSION: Biofilms that coalesce between the ribs of the contact case pose a threat because this area is difficult to thoroughly scrub and could act as a basis for infection through fouling of contact lenses. Of the four treatment regimens, microwave irradiation displayed the most consistent and highest rate of bacterial eradication. Tap water was less effective compared to microwave irradiation, and poses other harmful side effects, but greatly reduced CFU count compared to no treatment. MPS displayed the poorest bacterial eradication of the treatments. Thus, microwave irradiation is worth further investigation as a viable in-home disinfecting option.

Evolution of the Laser-Induced Spallation Technique in Film Adhesion Measurement.

Laser-induced spallation is a process in which a stress wave generated from a rapid, high-energy laser pulse initiates the ejection of surface material opposite the surface of laser impingement. Through knowledge of the stress-wave amplitude that causes film separation, the adhesion and interfacial properties of a film-on-substrate system are determined. Some advantages of the laser spallation technique are the noncontact loading, development of large stresses (on the order of GPa), and high strain rates, up to 108/s. The applicability to both relatively thick films, tens of microns, and thin films, tens of nm, make it a unique technique for a wide range of materials and applications. This review combines the available knowledge and experience in laser spallation, as a state-of-the-art measurement tool, in a comprehensive pedagogical publication for the first time. An historical review of adhesion measurement by the laser-induced spallation technique, from its inception in the 1970s through the present day, is provided. An overview of the technique together with the physics governing the laser-induced spallation process, including functions of the absorbing and confining materials, are also discussed. Special attention is given to applications of laser spallation as an adhesion quantification technique in metals, polymers, composites, ceramics, and biological films. A compendium of available experimental parameters is provided that summarizes key laser spallation experiments across these thin-film materials. This review concludes with a future outlook for the laser spallation technique, which approaches its semicentennial anniversary.

The effect of surface roughness on laser-induced stress wave propagation.

We investigate laser-induced acoustic wave propagation through smooth and roughened titanium-coated glass substrates. Acoustic waves are generated in a controlled manner via the laser spallation technique. Surface displacements are measured during stress wave loading by the alignment of a Michelson-type interferometer. A reflective coverslip panel facilitates capture of surface displacements during loading of as-received smooth and roughened specimens. Through interferometric experiments, we extract the substrate stress profile at each laser fluence (energy per area). The shape and amplitude of the substrate stress profile are analyzed at each laser fluence. Peak substrate stress is averaged and compared between smooth specimens with the reflective panel and rough specimens with the reflective panel. The reflective panel is necessary because the surface roughness of the rough specimens precludes in situ interferometry. Through these experiments, we determine that the surface roughness employed has no significant effect on substrate stress propagation and smooth substrates are an appropriate surrogate to determine stress wave loading amplitude of roughened surfaces less than 1.2 µm average roughness (Ra). No significant difference was observed when comparing the average peak amplitude and loading slope in the stress wave profile for the smooth and rough configurations at each fluence.

Biofilm rupture by laser-induced stress waves increases with loading amplitude, independent of location.

Integral to the production of safe and biocompatible medical devices is to determine the interfacial properties that affect or control strong biofilm adhesion. The laser spallation technique has recently emerged as an advantageous method to quantify biofilm adhesion across candidate biomedical surfaces. However, there is a possibility that membrane tension is a factor that contributes to the stress required to separate biofilm and substrate. In that case, the stress amplitude, controlled by laser fluence, that initiates biofilm rupture would vary systematically with location on the biofilm. Film rupture, also known as spallation, occurs when film material is ejected during stress wave loading. In order to determine effects of membrane tension on the laser spallation process, we present a protocol that measures spall size with increasing laser fluence (variable fluence) and with respect to distance from the biofilm centroid (iso-fluence). Streptococcus mutans biofilms on titanium substrates serve as our model system. A total of 185 biofilm loading locations are analyzed in this study. We demonstrate that biofilm spall size increases monotonically with laser fluence and apply our procedure to failure of non-biological films. In iso-fluence experiments, no correlation is found between biofilm spall size and loading location, thus providing evidence that membrane tension does not play a dominant role in biofilm adhesion measurements. We recommend our procedure as a straightforward method to determine membrane effects in the measurement of adhesion of biological films on substrate surfaces via the laser spallation technique.

Stress impacts sensory variability through cortical sensory activity motifs.

Medically unexplained symptoms in depression are common. These individual-specific complaints are often considered an 'idiom of distress', yet animal studies suggest that cortical sensory representations are flexible and influenced by spontaneous cortical activity. We hypothesized that stress would reveal activity dynamics in somatosensory cortex resulting in greater sensory-evoked response variability. Using millisecond resolution in vivo voltage sensitive dye (VSD) imaging in mouse neocortex, we characterized spontaneous regional depolarizations within limb and barrel regions of somatosensory cortex, or spontaneous sensory motifs, and their influence on sensory variability. Stress revealed an idiosyncratic increase in spontaneous sensory motifs that is normalized by selective serotonin reuptake inhibitor treatment. Spontaneous motif frequency is associated with increased variability in sensory-evoked responses, and we optogenetically demonstrate that regional depolarization in somatosensory cortex increases sensory-evoked variability for seconds. This reveals a putative circuit level target for changes in sensory processing and for unexplained physical complaints in stress-related psychopathology.

An Automated Home-Cage System to Assess Learning and Performance of a Skilled Motor Task in a Mouse Model of Huntington's Disease.

Behavioral testing is a critical step in assessing the validity of rodent models of neurodegenerative disease, as well as evaluating the efficacy of pharmacological interventions. In models of Huntington's disease (HD), a gradual progression of impairments is observed across ages, increasing the need for sensitive, high-throughput and longitudinal assessments. Recently, a number of automated systems have been developed to perform behavioral profiling of animals within their own home-cage, allowing for 24-h monitoring and minimizing experimenter interaction. However, as of yet, few of these have had functionality for the assessment of skilled motor learning, a relevant behavior for movement disorders such as HD. To address this, we assess a lever positioning task within the mouse home-cage. Animals first acquire a simple operant response, before moving to a second phase where they must learn to hold the lever for progressively longer in a rewarded position range. Testing with this paradigm has revealed the presence of distinct phenotypes in the YAC128 mouse model of HD at three early symptomatic time points. YAC128 mice at two months old, but not older, had a motor learning deficit when required to adapt their response to changes in task requirements. In contrast, six-month-old YAC128 mice had disruptions of normal circadian activity and displayed kinematic abnormalities during performance of the task, suggesting an impairment in motor control. This system holds promise for facilitating high throughput behavioral assessment of HD mouse models for preclinical therapeutic screening.