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Delamination of Bacterial Biofilms using Laser-Generated Shockwaves

  • Author(s): Ramaprasad, Vidyunmala
  • Advisor(s): Grundfest, Warren S
  • et al.
Abstract

The goal of this thesis is to explore the use of laser generated shockwaves (LGS) as a potential methodology to treat wound surfaces infected with bacterial biofilm as they impose a major financial, economic and social burden on the society.

First, the basics of LGS and how the concept could be used as a potential treatment technique needed to be understood. For this, LGS technique was initially used to delaminate biofilm grown on polystyrene and PDMS surfaces. The system is based on a Q-switched, Nd:YAG pulsed laser with an output wavelength of 1064 nm and a pulse width between 2-6 ns that ablates titanium-coated target (glass) under confinement, thereby generating unipolar compressive waves. The adhesion strength of S. epidermidis biofilms grown on polystyrene, under static growth conditions was found to be 22.75 MPa ± 0.16% using interferometry techniques. Biofilm material properties measured by other techniques vary significantly due to the viscoelastic effect of biofilms under low strain rates. LGS technique exclusively uses compressive waves and high strain rate loading, thus the decohesive failure observed is an intrinsic strength measurement of the material and does not vary.

A low-cost, high-speed imaging system was developed and characterized to capture the LGS as they travel through the titanium- coated target and coupling medium. Cavitation bubbles forming at the glass/ water interface, and propagating in the same direction as the compressive wave were successfully imaged. As observed during biofilm delamination experiments these bubbles were responsible for causing localized delamination of the biofilm.

Preliminary studies to examine the safety of LGS on ex vivo skin (porcine skin) were conducted. These studies were conducted to ascertain the impact of LGS on skin tissues that would underlie the biofilm in a typical wound bed. LGS on ex vivo porcine skin caused no qualitative structural damage at energy fluences between 16.68 and 70.42 mJ/mm2. All the 3 layers of the skin, the stratum corneum, epidermis and dermis remained intact. The collagen structure also appeared undisturbed by LGS treatment.

A thorough study to determine the damage threshold for ex vivo porcine skin was carried out. The effect of a wide range of energy fluences from 250 mJ (35.38 mJ/mm2) to 1500mJ (212.28 mJ/mm2) was studied. A large number of samples (total of 105 tissue samples) was used in this study. By scoring the tissue samples based on the damage that had occurred to them and performing statistical analysis on the scores, the damage threshold for ex vivo porcine skin was determined to be 1500mJ (212.28 mJ/mm2).

The biofilm delamination studies and experiments to determine damage threshold of ex vivo porcine skin has provided an optimal range of energy fluences that would cause biofilm delamination as well as be safe to use for in vivo experiments.

Finally, the LGS propagation through the coupling medium, biofilm and underlying muscle was modeled using COMSOL. This model would help predict the outcome of changing any of the parameters in the experimental set up. It will also be able to predict the effect of multiple shockwaves on the biofilm and the underlying muscle tissue. This will drastically reduce the number experimental trials needed to be performed when any changes to the system are proposed in order to make the system more suitable for in vivo or clinical settings saving tremendous amount of money, time and effort.

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