<p>Intervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.</p><p>To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.</p><p>NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.</p><p>The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.</p> / Dissertation

Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth. At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies. Next, we sought to apply the developed imaging techniques, along with other complementary methodologies, to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling. In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.

In this study a microbial polyester, poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and its blends were wet or electrospun into fibrous scaffolds for tissue engineering. Wet spun fiber diameters were in the low micrometer range (10-50 &amp / #956 / m). The polymer concentration and the stirring rate affected the properties the most. The optimum concentration was determined as 15% (w/v). Electrospun fiber diameters, however, were thinner. Solution viscosity, potential, distance between the syringe tip and the collector, and polymer type affected the morphology and the thickness of beads formed on the fibers. Concentration was highly influential / as it increased from 5% to 15% (w/v) fiber diameter increased from 284 &plusmn / 133 nm to 2200 &plusmn / 716 nm. Increase in potential (from 20 to 50 kV) did not lead to the expected decrease in fiber diameter. The blends of PHBV8 with lactide-based v polymers (PLLA, P(L,DL-LA) and PLGA (50:50)) led to fibers with less beads and more uniform thickness. In vitro studies using human osteosarcoma cells (SaOs-2) revealed that wet spun fibers were unsuitable because the cells did not spread on them while all the electrospun scaffolds promoted cell growth and penetration. The surface porosities for PHBV10, PHBV15, PHBV-PLLA, PHBV-PLGA (50:50) and PHBV-P(L,DL)LA were 38.0&plusmn / 3.8, 40.1&plusmn / 8.5, 53.8&plusmn / 4.2, 50.0&plusmn / 4.2 and 30.8&plusmn / 2.7%, respectively. Surface modification with oxygen plasma treatment slightly improved the cell proliferation rates. Consequently, all scaffolds prepared by electrospinning revealed a significant potential for use in bone tissue engineering applications / PHBV-PLLA blend appeared to yield the best results.

Aortic valve disease is among the third most common cardiovascular disease worldwide, and is also a strong predictor for other cardiac related deaths. Altered mechanical forces are believed to cause changes in aortic valve biosynthetic activity, eventually leading to valve disease, however little is known about the cellular and molecular events involved in these processes. To gain a fundamental understanding into aortic valve disease mechanobiology, an ex vivo experimental model was used to study the effects of normal and elevated cyclic stretch on aortic valve remodeling and degenerative disease. The hypothesis of this proposal was that elevated cyclic stretch will result in increased expression of markers related to degenerative valve disease. Three aspects of aortic valve disease were studied: (i) Altered extracellular matrix remodeling; (ii) Aortic Valve Calcification; and (iii) Serotonin-induced valvulopathy. Results showed that elevated stretch resulted in increased matrix remodeling and calcification via a bone morphogenic protein-dependent pathway. In addition, elevated stretch and serotonin resulted in increased collagen biosynthesis and tissue stiffness via a serotonin-2A receptor-mediated pathway. This work adds to current knowledge on aortic valve disease mechanisms, and could pave the way for the development of novel treatments for valve disease and for the design of tissue engineered valve constructs.

The evolution of biomechanical behavior of arteries plays a key role in the onset and progression of cardiovascular disease. Biomechanical behavior is governed by the content and organization of the key structural constituents (e.g., collagen, elastin, and smooth muscle) and vessel geometry. The evolution of biomechanical behavior of arteries is governed by biologically-mediated synthesis, degradation, and reorganization of these key structural constituents. A hallmark goal in biomechanics is quantifying the relationship between the microstructure of tissues and their mechanical response throughout tissue growth and remodeling; this will provide a crucial link in understanding the tissue level effects of biological processes involved in disease and normal growth Fibulin-5 (fbln5) is an ECM protein that binds tropoelastin and interacts with integrins. Arteries from fbln5 knockout mice lack functional elastic fibers and provide a system for investigating the link between an artery's microstructure and its mechanical response. The overall goal of this project was to develop multi-scaled theoretical and experimental frameworks to quantify the relationship between microstructural content and organization and tissue level material properties of arteries from fbln5 null mice and littermate controls and to quantify the effects of fbln5 on the in vivo maturation of mouse carotid arteries. We found significant differences in the mechanical properties of carotid arteries of fbln5 null mice, and these differences were correlated with altered extracellular matrix organization. We also developed a microstructurally-motivated 3-dimensional constrained mixture model for vascular growth and remodeling. Using physiological rates of constituent growth and turnover, the model captured the salient findings found in the literature. Incorporating experimentally measured fiber angle data into constitutive relations yielded greater predictive accuracy. This dissertation incorporates experimental data quantified at the micro (microstructural-level fiber distributions) and macro (tissue-level mechanical response) scale and incorporates these data into microstructurally motivated constitutive relations. The use of structurally motivated constitutive relations and experimentally measured microstructural data provides a foundation for future work in further understanding the relationship between processes governing microstructure and the tissue level effects of disease and normal growth.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Anatomy and Neurobiology. Title from title-page of electronic thesis. Bibliography: leaves 181 - 202. Available online via the Internet.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 91 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

The Drosophila dorsal group gene pipe provides the crucial link that transmits dorsal-ventral (DV) polarity information from the ovary to the embryo. Females homozygous for mutations in pipe produce dorsalized embryos. pipe encodes ten protein isoforms with amino acid sequence similarity to vertebrate glycosaminoglycan 2-O-sulfotransferases, suggesting that Pipe functions by modifying a carbohydrate-bearing molecule that controls embryonic DV patterning. Two major components of my project have been to examine the functional specificities of different Pipe isoforms and to identify Pipe's enzymatic substrate and learn how it participates in DV pattern formation. I have used two approaches to investigate whether the various Pipe isoforms share the same functional specificities. In one approach, I expressed each isoform in the follicle cells and found that the expression of only one of them was able to rescue the pipe mutant phenotype or ventralize progeny embryos. In a second set of transgenic studies, three of the other isoforms were individually shown to restore the production of a pipe-dependent sulfated epitope when expressed in the salivary glands of otherwise pipe null mutant embryos. These data indicate that distinct functional specificities are associated with the various Pipe protein isoforms. In addition, these studies allowed me to determine that embryos from females lacking endogenous pipe expression nevertheless retain polarity along their dorsal-ventral axis, suggesting the existence of a second polarizing signal in addition to the ventral transcription of pipe. To identify Pipe’s substrate, I developed a technique for metabolic labeling which enabled me to identify a molecule exhibiting Pipe-dependent sulfation. This molecule was identified as the protein Vitelline Membrane-Like (VML), a putative component of the vitelline membrane layer of the eggshell. The involvement of VML in dorsalventral patterning was demonstrated on the basis of the enhancing effects of a vml mutation on the severity of dorsalization of embryos from females of a sensitized genetic background. Thus, VML represents a bona fide substrate of Pipe that participates in the establishment of dorsal-ventral polarity. In these studies I was also able to show Pipedependent sulfation of other vitelline membrane components which may also influence embryonic dorsal-ventral patterning. / text