Cell- and Extracellular Matrix-Based Approaches to Investigate Diabetic Fibroblasts and Improve Wound Healing
Date of Award
Doctor of Philosophy (PhD)
Biomedical Engineering (ENAS)
Chronic wounds are a major complication associated with diabetes. Often presented as foot ulcers, diabetic chronic wounds have a 5-7% prevalence among patients and can lead to serious consequences such as sepsis and amputation. Despite the devastating impact of such complications on patients’ health, current clinical interventions have limited efficacy, in part, due to 1) a limited understanding of the underlying cellular mechanism and 2) the lack of effective engineering solutions that can improve therapy. This thesis addresses these two issues by first investigating dysfunctions and altered molecular mechanisms in diabetic fibroblasts and then creating a novel, decellularized biomaterial enriched with growth factors to accelerate wound healing. Fibroblasts are a major cell population that participate in the wound healing process. In response to injury, they proliferate and migrate into the wound space, playing a role in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were deployed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived ECM (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblast. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were partly due to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the Rac1-WAVE2-actin axis, and illuminating a new function of TSP2 in regulating cytoskeleton organization. Next, a novel approach of manipulating cell lines to produce a growth factor-enriched biomaterial was demonstrated and tested both in vitro and in vivo. By combining transfection of high-affinity platelet derived growth factor (PDGF-PIGF) with standard decellularization techniques, we demonstrate the feasibility of rapidly engineering CDM. Subsequent in vitro studies revealed a strong retention of PDGF within the CDM, facilitating cell proliferation and sustained migration. When applied to full-thickness skin wound, this novel material resulted in better wound resolution after two weeks, as shown by earlier infiltration of macrophages, improved angiogenesis, increased fibroblast recruitment, decreased wound width, and reduced epithelial thickness. Overall, this thesis examines the functional impairments of diabetic fibroblasts and identifies Rac1-WAVE2 as the key regulatory axis that is disrupted in diabetes. Concurrently, a previously unknown role of TSP2 in regulating cytoskeleton organization is discovered. This work also provides a novel engineering approach to manufacture CDM-based biomaterials that can be easily modified to provide effective wound treatments.
Xing, Hao, "Cell- and Extracellular Matrix-Based Approaches to Investigate Diabetic Fibroblasts and Improve Wound Healing" (2022). Yale Graduate School of Arts and Sciences Dissertations. 537.