Dissertation
Integration of magnetic tweezers and traction force microscopy to investigate extracellular matrix microrheology and keratinocyte mechanobiology
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2022
DOI: 10.25820/etd.006783
Abstract
In humans, autoimmune bullous diseases (AIBDs) represent several distinct types of severe acquired autoantibody-mediated conditions causing blisters, erosions, and ulcerations of the skin and mucosa leading to significant morbidity (e.g., infections, scarring) and mortality. In these conditions, specific cell-cell and cell-extracellular matrix (ECM) anchoring junctions within the complex cytoarchitecture of the epidermis are rendered dysfunctional. In AIBDs such as pemphigus vulgaris (PV) and bullous pemphigoid (BP), only impairment of desmosome or hemidesmosome function is known to cause mechanical skin fragility, whereby these junctions fail to connect to keratin intermediate filaments or between a keratinocyte and its adjacent extracellular matrix, respectively. Intriguingly, there are no known AIBDs in which pathogenic antibodies target adherens junctions or focal adhesions connected to microfilaments within. While the targets of pathogenic autoantibodies have been identified in PV and BP, the biophysical mechanisms underlying the mechanical disintegration of the epidermis following autoantibody exposure remain mostly unknown. Therefore, current treatment regimens are based on corticosteroids and/or adjuvant steroid-sparing immunosuppressants that aim to quell autoantibody production and inflammation, where long-term use of either carries a substantial risk of serious events such as infection and osteoporosis, especially for the elderly.
To bridge mechanistic knowledge gaps in our understanding of AIBDs, new experimental approaches are needed. To date, fundamental studies of force transmission within multicellular constructs of human epidermal keratinocytes or between keratinocytes and dermal mimics have been lacking. To optimally explore force transmission within a multicellular keratinocyte sheet in vitro, the experimenter should have the ability to apply a force that is (1) specific to an isolated anchoring junction of interest and (2) of sufficient magnitude to generate cellular and/or substrate deformations that can be physically resolved and characterized within the framework of applied mechanics. Based on these constraints, the primary objective of this work is to develop and characterize a new experimental methodology that integrates the well-established techniques of magnetic tweezers (MTs) and traction force microscopy (TFM) to be used as a basis for future advanced investigations of human epidermal keratinocyte mechanobiology.
During this work, many technical advances were realized, including the design and construction of an innovative MT device operating with magnetic flux density feedback control, the adaptation of TFM to handle dynamic experiments with exceptionally large spatiotemporal datasets, and the development of new keratinocyte culture methods compatible with MT-TFM instrumentation. The potential utility of the integrated MT TFM methodology for use in studies related to keratinocyte microbiology and extracellular matrix microrheology was validated with two proof-of-concept applications: (1) investigation of the local mechanical properties of a type I collagen gel reconstituted in vitro, and (2) demonstration of how a force applied to a focal adhesion contact on the apical surface an isolated keratinocyte generates observable deformations of the underlying collagen substrate. Together, the two model experiments detailed in this work suggest that the methodology of integrated MT TFM will be a useful tool in future investigations that attempt to increase our understanding of the biophysical mechanisms underlying tissue damage that arises in the context of AIBDs. We hope that such new knowledge will provide an impetus for the development of new innovative therapeutics that are designed to strengthen the structural integrity of the epidermis –– a dramatically different approach compared to current immunosuppressive treatments that target the inhibition of autoantibody production and the resultant inflammatory response.
Details
- Title: Subtitle
- Integration of magnetic tweezers and traction force microscopy to investigate extracellular matrix microrheology and keratinocyte mechanobiology
- Creators
- Waddah Ibrahim Moghram
- Contributors
- John C. Selby (Advisor)Edward A. Sander (Committee Member)James A. Ankrum (Committee Member)Madhavan L. Raghavan (Committee Member)Eric E. Nuxoll (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Engineering
- Date degree season
- Spring 2022
- DOI
- 10.25820/etd.006783
- Publisher
- University of Iowa
- Number of pages
- xxviii, 238 pages
- Copyright
- Copyright 2022 Waddah Ibrahim Moghram
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Date submitted
- 04/26/2022
- Date approved
- 05/11/2022
- Description illustrations
- Illustrations, charts, graphs, tables
- Description bibliographic
- Includes bibliographical references (pages 171-189).
- Public Abstract (ETD)
In humans, autoimmune bullous diseases (AIBDs) represent a class of chronic and severe acquired skin diseases where the body produces autoantibodies triggering blisters and ulcers in the skin and mucosa. Loss of the mechanical barrier function of skin and mucosa over widespread areas can subsequently lead to opportunistic infections, and in some instances, even death. While the anchoring junctions of epidermal keratinocytes targeted by pathogenic autoantibodies are known for many types of AIBDs, the biophysical mechanisms causing the breakdown of these anchoring junctions remain mostly unknown. Thus, current treatments rely on steroids or immunosuppressants to suppress antibody production and reduce inflammation.
To bridge the mechanistic knowledge gaps of AIBDs, new experimental approaches are needed. To date, there are few fundamental studies of force transmission between epidermal keratinocytes or between keratinocytes and neighboring extracellular matrix. To meet this goal, we developed a new experimental method integrating the well-established techniques of magnetic tweezers (MTs) and traction force microscopy (TFM). The integrated methodology was validated with two proof-of-concept model experiments. The first was where we measured the local mechanical properties of type I collagen, and the second was where we studied force transmission between a keratinocyte and its underlying culture matrix. Together, these two demonstrations suggest that the integrated MT-TFM method will be a useful tool to better understand the missing mechanistic knowledge of AIBDs. Moreover, it is our hope this can start us on the path of developing innovative therapeutics, especially ones designed to strengthen the structural integrity of the epidermis instead of the current adverse immunosuppressive treatments.
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering
- Record Identifier
- 9984422459702771
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