Collagen I, a natural protein found in animal tissues, can self-assemble into fibrous matrices that support cell and tissue growth. Peptide mimics of collagen are able to recapitulate this selfassembly process towards the development of biomaterials for tissue engineering. In recent years, the metal mediated self-assembly of collagen mimetic peptides (CMPs) has allowed access to various particle morphologies. Herein, two studies are presented. In the first, NCOH-FOGER, a cell adhesive CMP capable of metal-triggered self-assembly, was utilized to develop a model system to mimic natural collagen’s interactions with endothelial cells. Notably, a cobalt(III)- NCoH-FOGER assembly was able to induce endothelial cells to form network-like structures. In the second, a CMP was modified to include an unnatural amino acid, L-4-trans-fluoroproline, which increased the thermostability of its folded state. The effect of this substitution on the morphology of self-assembled particles was evaluated.

The extracellular matrix (ECM), as the native cellular substrate, provides necessary mechanical and biological signals to cells. Cells exert forces in the nanonewton range, which when applied over time can strain extracellular matrix fibers until breakage. Cells and tissues inherently interact mechanically with their surrounding matrix, so tissue engineering materials would benefit from the ability to fully exploit mechanical-biochemical interactions to enhance integration with the human body. In this work, I developed an increased understanding of ECM fiber mechanical and mechano-biochemical properties. First, I generated novel composite ECM fibers that can be used to study combinations of ECM proteins in a controlled way. I determined how protein composition impacts mechanical properties of novel single ECM fibers in a hydrated state and showed how mechanical properties can be tuned through composition. Next, I assayed for strain and heparin-sensitive allosteric binding of ligands to fibronectin and fibrin fibers, and determined that the binding of two key growth factors is impacted by strain and heparin. Finally, I investigated the impact of fiber strain, heparin-pretreatment, and growth factor interactions on endothelial cell migration. The novel contributions of this project are the generation of new composite extracellular matrix fiber types with tunable mechanical properties, as well as the identification of extracellular matrix protein mechanosensitive and heparin-sensitive interactions with growth factors and their impact on endothelial cell migration, which could be used to aid in the design of protein-based biomaterials for cardiovascular applications. / 2024-05-23T00:00:00Z

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Basic research into the histology, endocrine control, and local regulation of prepuberal ruminant mammogenesis was conducted to provide a better understanding of this important developmental period. Histologic features of prepuberal ruminant mammary parenchymal morphogenesis were examined in tissue samples taken from ewe lambs at 2 (n = 5), 3 (n = 15), 6 (n = 26), 9 (n = 7), 12 (n = 5), and 13 wk (n = 20), and from Holstein heifers at 4 (n = 1) and 6 mo (n = 2). Examination of approximately 8000 histologic sections revealed that mammary parenchymal morphogenesis in sheep and cattle occurs through the proliferation of highly arborescent ductal structures embedded in a dense stroma. These observations contrast strongly with models of mammogenesis based on murine mammary development. The formation of luminal spaces and the expansion of ducts also differed from murine mammogenesis models. Luminal spaces were shown to develop through a progressive separation of opposing sides in initially solid ductal structures. Likewise, our investigation of prepubertal ovine mammogenesis revealed that parenchymal weight, 3H-thymidine labeling, stromal weight, and parenchymal DNA were all unaffected by ovariectomy (P > 0.05), in marked contrast to the dramatic reduction in mammary development following ovariectomy in rats, mice, and heifers. Responsiveness to exogenous estrogen (0.1 mg/kg) was demonstrated by increased 3H-thymidine labeling (P < 0.05) in both intact and ovariectomized lambs. Three dimensional collagen gel cultures of bovine mammary organoids from the peripheral (OUTER) and medial (INNER) parenchymal zones were used to characterize the proliferative and morphogenetic responses to local-acting growth factors. The proliferation of OUTER cells was 2 to 3 times greater than INNER cells (P < 0.0001) in response to IGF-I stimulation. Dramatic differences in the morphology of INNER and OUTER organoids were also observed. INNER cells grew into smooth-edged colonies when treated with heifer serum but stellate colonies when treated with other mitogens. OUTER cells grew into stellate colonies regardless of mitogen treatment. These investigations highlight the fact that a great deal more research into the basic physiology of prepuberal ruminant mammogenesis is required and that dogma developed in murine model systems may not be applicable to ruminant mammary physiology. / Ph. D.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The ultrastructural mechanism for strain rate sensitivity of collagenous tissue has not been well studied at the collagen fibril level. The objective is to reveal the mechanistic contribution of the collagen fibril to strain rate sensitivity. Collagen fibrils underwent significantly greater fibril strain relative to global tissue strain at higher strain rates. A better understanding of tendon mechanisms at lower hierarchical levels would help establish a basis for future development of constitutive models and assist in tissue replacement design. High rate mechanical property of tendon was also studied. Tendon was compressed under high strain rate (550 /s) using a polycarbonate split Hopkinson pressure bar (PSHPB). The objectives are to investigate the tissue behavior of porcine tendon at high rates. Tendon’s high rate behavior was compared with brain and liver at both hydrated and dehydrated states to investigate how water content and ultrastructural affect high rate responses of soft tissues.

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