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A Nanoscale Investigation of Pathogenic Microbial Adhesion in Biomaterial Systems

Microbial infections of medical implants occur in 10% of the more than 20 million surgical procedures carried out annually in the United States. The additional treatments required to address these infections generate more than $11 billion in additional patient costs, increase recovery time, and decrease overall patient quality of life. As the population ages, the number of necessary and voluntary surgical procedures increases; The rate of infection increases proportionately. While treatments are available, the biofilm mode of growth confers resistance to antimicrobial therapies up to 500 times greater than that of planktonic microbes. Currently, the only guaranteed method of removing an established microbial implant infection is through surgical excision of the implant and surrounding tissues. While removing the original infection, additional colonization and pathogenesis may take place. This research explores the a priori assumption that a medical implant infection cannot occur unless a microbial cell is capable of adhering to the implant surface. From that assumption, the following sections will focus primarily on identifying the necessary and sufficient factors influencing microbial adhesion, discretizing those factors into measurable quantities, and developing methods by which those factors may be mitigated or eliminated. Following is a brief summary of each major topic treated within this research period. Development of a Benchmark System: We have characterized the interactions between Pseudomonas aeruginosa ATCC 10145 and Candida parapsilosis ATCC 90018 using a novel method of cellular immobilization, which emphasizes minimal chemical modification of the cell surface. This research describes the very different force-separation interactions seen between C. parapsilosis and both a common medical implant material (viz., silicone rubber) and a nascent P. aeruginosa biofilm grown on the same material. This study was the first step in developing an ab initio technique which may be used to determine the relative affinity of a microbial cell for an implant material surface. The Role of the Substrate: Microbial adhesion to a medical implant device involves two major components, being the microbe itself, and the substrate to which it adheres. Each of the two has specific and unique surface chemical and textural characteristics which, when combined, allow for microbial colonization and subsequent infection. The goal of this study was to identify correlations between the adhesive strength of Staphylococcus epidermidis to a variety of chemically and texturally distinct substrates, and common surface characterization parameters (e.g., surface roughness and water contact angle). Relationships to adhesive strength did not demonstrate statistically significant or consistent trends. To extend upon the correlation parameters, we have employed a Discrete Bonding Model, which characterizes the surface texture according to Mandelbrot fractal theory. Correlations between the adhesive strength and the observational scale show stronger relationships, indicating a significant contribution of the surface texture to a microbe's ability to colonize a surface. Finding a Surface That Cannot Be Touched: Historically, AFM force-separation curves demonstrating only repulsive behavior on extension of the piezoactuator have been largely ignored, in terms of quantitative modeling of the interactions. In bacterial systems, such behavior describes the majority of the force profiles recorded by the instrument. As a result of the former lack of study, the latter data sets have remained unanalyzed and unanalyzable. Building on existing mathematical models, we have developed an analytical method by which the point of zero separation between a surface (viz., the microbial cell wall) coated with a polymer brush and an AFM probe may be quantitatively identified

Microbial Adhesion to Medical Implant Materials: An Atomic Force Microscopy Study

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an Atomic Force Microscope (AFM) cantilever, and a biomaterial or biofilm substrate. Distinct adhesive interactions exist between Candida parapsilosis and both unmodified silicone rubber and Pseudomonas aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive interactions with magnitude of 2.3 ± 0.5 nN (SD) in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force increases to 3.5 ± 0.75 nN (SD), and is preceded by a 2.5 nN repulsion at approximately 175 nm from the cell surface. This repulsion may be attributed to steric and electrostatic interactions between the two microbial polymer brushes. Young's moduli for microbes and biofilms were calculated using Hertzian contact models. These produced values of 0.21 ± 0.003 MPa (SD) for the C. parapsilosis-silicone rubber system, and 0.84 ± 0.015 MPa (SD) for the C. parapsilosis-biofilm system. This technique may be extended to calculate the work per unit contact area involved in the attractions in experimental data. For example, the work of adhesion using a spore probe is an order of magnitude greater for unmodified silicone rubber than for a P. aeruginosa biofilm. This indicates a high affinity for silicone rubber, and suggests that this material is vulnerable to infection by C. parapsilosis in vivo. We have also demonstrated that AFM force curve analysis using established qualitative and quantitative models fails to accurately represent the physical interactions taking place between the probe and sample for the case where a polymer brush exists on the substrate, the probe, or both. As such, an approximate method defining the sample surface as the actual surface plus some vertical dimension associated with the maximum compressible thickness of the polymer brush is discussed. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative evaluation of the materials used for medical implantation. It also provides a link between the physicochemical and physicomechanical properties of these materials and the nanoscale interactions leading to microbial colonization and infection. The goal of this research is to study this link and determine how best to exploit it to prevent microbial infections of medical implant materials.</p

Civil War studies on Martha's Vineyard.

Martha's Vineyard was a significant source of aid to the Union during the United States Civil War. Working with the Martha's Vineyard Historical Society, the group examined Bellum artifacts, transcribed letters from an Edgartown soldier, and provided suggestions for a future exhibit regarding Martha's Vineyard's participation in the Civil War. From this work, the people of the present will more easily see the role played by Martha's Vineyard in the largest war to take place on the North American continent

Freeform production of cartilage structures using aerosol methods

This project dealt with the construction of freeform structures embedded with cartilage cells. Cells were delivered in 18 wt% Pluronic? solution via a pressure atomizer onto a heated surface. The structure was constructed by adding subsequent layers after drying. Structures were analyzed for cell viability, total cell count and axial cell distribution using Trypan blue dye. We determined that 67.7% of total cells survived the process and that they were evenly distributed throughout the structure