Mechanical properties of single keloid and normal skin fibroblast measured using an atomic force microscope.

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Abstract

The human body consists of a number of very complex, highly specialised organs which perform a variety of functions that are essential to life and health. One of the main functions of the skin, the largest of the human organs, is to maintain the integrity of the body. It does this by acting as a physical barrier, preventing micro-organisms and other potentially harmful substances from entering the body. When the integrity of the skin is damaged through injury, a self-protective mechanism is triggered and the reparative wound healing process begins. Under normal circumstances the wound healing process culminates in the skin recuperating its normal characteristics and functions at the site of the injury, with only a small visible mark being left behind. However, in some cases the wound healing process may become altered leading to the production of abnormal scars, such as keloids. Keloid scars are formed from scar tissue at the site of an injury, as a result of excessive tissue repair that extends beyond the boundaries of the original wound. These scars are characterised by excess collagen deposition produced during the wound healing process. It is estimated that as many as 20% of the black and Hispanic population are affected by keloid scarring. In addition to the aesthetic aspect, keloid scars can also be painful, itchy and prone to become infected. Keloid scar formation can be triggered by skin injuries caused by, for example, acne, wounds, shaving, burns, and surgical incisions. The mechanism by which keloid scars form is currently not well understood and consequently no effective treatments exist to date.This thesis describes an investigation into the mechanical properties of single keloid and normal skin fibroblast cells for the purpose of establishing if there is a quantitative difference between the two types of cells. This information will be of benefit to researchers looking for a better understanding of the keloid formation mechanism and for those seeking improved treatments. An atomic force microscope (AFM) was employed to indent single Keloid and normal skin fibroblast cells taken from five patients. Values for the apparent Young’s modulus of the cells were then calculated by fitting the experimental data using Hertz’s model. Apparent Young’s modulus values were then compared. The findings of the analysis indicate that statistically, there is a significant difference in the Young’s modulus values of normal and keloid cells, with keloid cells exhibiting substantially greater stiffness than normal skin fibroblast cells. To enable the keloid and normal skin fibroblast cells to be studied in as close to their natural, physiological environment as is possible the AFM experiments described in this thesis were undertaken in a phosphate buffered saline (PBS) solution. In such cases the use of a fluid medium presents additional complexities, not least of which is the introduction of a hydrodynamic drag force due to viscous friction of the cantilever with the liquid which can affect the experimental data obtained and consequently any material properties calculated as a result of using these data. In order to investigate the effect of dragging force on the experimental data obtained from the AFM a novel integrated finite element based model was developed. The model, described in this thesis, provides quantification of the drag force in AFM measurements of soft specimens in fluids, consequently enabling more accurate interpretation of the data obtained from AFM experimentation. The model is validated using extensive data obtained from AFM experimentation undertaken in a number of fluids of different viscosities, at a variety of tip velocities and platform-tip separations and by comparison with an existing analytical model. The novel model is shown to accurately account for drag forces in AFM in fluid media without the need for extrapolation of experimental data and can be employed over the range of tip geometries and velocities typically utilised in AFM experimentation.The work described in this thesis demonstrates that the AFM is a valuable tool that can be used to successfully investigate the mechanical properties of biological samples in fluids. It was shown that increased accuracy in the interpretation of data obtained from AFM experimentation can be obtained by taking into account the hydrodynamic drag force due to viscous friction of the cantilever with the liquid. The investigation into the mechanical behaviour of keloid cells described in this thesis significantly adds to the yet small body of research undertaken on keloid cells to date. The findings of the investigation will provide valuable information that will be of benefit in the future to researchers looking to develop effective treatments for the prevention, reduction or removal of keloid scars.

Keyword(s)

AFM, Keloid scar, keloid cell, fibroblast, Drag force, FEM

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