DOI

10.17077/etd.ocfoz0gh

Document Type

Dissertation

Date of Degree

Summer 2017

Access Restrictions

.

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biomedical Engineering

First Advisor

Edward A. Sander

First Committee Member

Michael Mackey

Second Committee Member

John C Selby

Third Committee Member

M.L. Raghavan

Fourth Committee Member

James Ankrum

Abstract

Wound healing is an intrinsic response to injury or disease that generally results in scarring. In skin, restoration of the barrier function after wounding is critically dependent on re-epithelialization. During re-epithelialization keratinocytes from the wound margin migrate over the wound bed, proliferate and re-differentiate to make an intact epidermis. Mechanical cues may play an important role in epidermal sheet formation and re-epithelization.

Here, polyacrylamide (PA) gels with tunable stiffness were used to study the potential contribution of mechanical properties of the wound bed in re-epithelialization. Live cell imaging and deformation tracking microscopy was performed on primary human keratinocytes maintained on soft (1.2 kPa) and stiff (24 kPa) PA gel substrates. The results of this study indicated that the formation of keratinocyte aggregates was significantly different on soft versus stiff polyacrylamide gels, with smaller spread contact area, increased migration velocities, increased rates of aggregate formation, and more cells per aggregate for keratinocytes cultured on soft gels versus stiff gels, respectively. The differences may be due to cell–cell mechanical signaling generated via local substrate deformations in the underlying substrate. These deformations were substantially larger for soft gels.

Broad-spectrum proteomics was performed to investigate which proteins were expressed differentially on soft and stiff PA gels. Protein lysates from soft and stiff samples were analyzed by LC-MS/MS (Q-Exactive). Expression of 56 proteins differed significantly between keratinocytes on soft and stiff substrate samples. The presence of serotransferrin, one of the most prominent protein candidate presented higher in the soft samples, was confirmed using western blot. Further analysis needs to be conducted to investigate the rest of protein candidate in proteomics results.

The PA gel system was then used to explore the role that keratin intermediate filaments play in mechanosensing and force generation. Knock-out mouse keratinocytes that were missing certain keratins and their corresponding wild type controls demonstrated differences. Knock-outs were less spread out, had impaired actin formation, could not deform the substrate, and hardly made aggregates on soft substrates. These results revealed the importance of keratin intermediate filaments in keratinocyte mechanobiology.

In order to characterize keratinocyte mechanosensing, a needle was used to make controlled local mechanical deformations in soft PA gels in a defined distance from isolated single cells. Keratinocytes responded to needle-induced substrate displacements by changing direction, and migrating towards the needle. The inhibitors, Y27632 and blebbistatin were used to inhibit Rho kinase and myosin phosphorylation. Both Y27632 and blebbistatin impaired directed migration toward the needle.

Together my results reveal new insights on keratinocytes mechanobiology, which could help in the development of novel healing strategies.

Pages

xx, 148 pages

Bibliography

Includes bibliographical references (pages 121-130).

Copyright

Copyright © 2017 Hoda Zarkoob

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