Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by pro... more Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.
The development of hard tissues such as bone and teeth are examples of biomineralization processe... more The development of hard tissues such as bone and teeth are examples of biomineralization processes in which organisms actively direct mineralization. The formation of hard tissues is a highly regulated process involving the coordination of extracellular matrix production by the cells and the mineralization of the matrix network regulated by proteins secreted by the cells. The formation of these tissues follows a sequence of events in a spatial and temporal manner dictated by cellular processes. In addition to the cells involved in the formation and remodeling of hard tissues, such as osteoblasts (OBs) and osteoclasts (OCs) in bone, other cell-types, such as breast and prostate tumor cells, can interact with bone. Two strategies were taken in this thesis to understand how cells are involved in the mineralization process. Cell culture systems were designed to examine cell-mineral interactions or to examine cell-mediated mineral formation. Chapter 1 discusses the requirements for sustaining cell viability and function as well as the various components involved in designing cell culture systems such as cell source, culture method, and mineral source. Culture systems to study cell-mineral interactions require an environment containing the mineral of interest for cells to be cultured upon. Hydroxyapatite (Ca 10 (PO 4) 6 (OH) 2 , HA) is the main mineral component of bone and has been noted to change in size, shape, and crystallinity with age, anatomical location, and disease progression. Chapter 2 describes a two-step method for synthesizing hydroxyapatite nanoparticles with controlled shape, size, and crystallinity in order to evaluate how differences in HA crystal properties can affect cellular response. Three culture systems were developed and described in this work. Two systems were developed to utilize the synthetic hydroxyapatite nanoparticles (prepared in Chap. 2) for examining cell-mineral interactions. In Chapter 3, dry-annealed synthetic and commercial HA particles were combined with polylactide(co-glycolide) (PLG) and spin-coated onto glass substrates to form a film of PLG-HA to examine how changes in crystallinity can affect OB attachment and matrix production. Chapter 4 describes the integration of a series of hydrothermally aged HA nanoparticles into porous PLG scaffolds to examine breast cancer cell response to various HA nanoparticles with discrete sizes. Finally, Chapter 5 details the third system designed for culturing cells in a 3-D gel system that allows for the presence of 1-D gradients to study cell-mediated mineral formation with articular chondrocytes. Due to the breadth of components involved, designing culture systems for mineralization studies are complex and selection of cell source, culture method, and mineral source should be dictated by research questions of interest of particular aspects of these processes (Chapter 1). Chapter 2 demonstrates how controlling for reactant salts, reactant concentration, reaction duration, and reaction volume in the initial precipitation step leads to changes in shape (e.g., rounded to plate-like) and phase (e.g., brushite to HA) while further post-processing techniques can be applied to tune phase, size, crystallinity, and dispersity. Surfaces containing dry-annealed particles increased fibronectin secretion from osteoblasts in Chapter 3, however this response was not due solely to an increase in crystallinity, and other changes to the surfaces due to heat treatment need to be examined. Breast tumor cells in Chapter 4 attached better on scaffolds containing smaller, less crystalline HA, however interleukin-8 (IL-8) secretion was upregulated in cells cultured on scaffolds containing larger, more crystalline HA, indicating IL-8 as a potential factor in facilitating breast cancer tumor metastasis processes. Chapter 5 establishes a 3-D culture system which can support cell viability and matrix production with future work to optimize the system for culturing cells under mineralizing gradients. iii BIOGRAPHICAL SKETCH Debra DengWen Lin was born on September 4, 1985 to TsanHai Lin and ShuFen Huang. Her parents had immigrated to the United States in search of better opportunities and encouraged Debra to do the same. She found science with medical applications to be fascinating and sought colleges with research opportunities in the field. Debra graduated from Auburn High School ranked number 1, varsity tennis MVP, gold medalist pianist, with a bunch of medals from Science Olympiad, and went on to attend the Massachusetts Institute of Technology fall of 2003. At MIT, Debra joined Course 3, the Department of Materials Science & Engineering. During that time, she authored a patent at her summer internship at Toyota Motor Corporation in Japan, pulled an all-nighter with a group of friends to write a successful SBIR grant, and drove around Boston on the MIT ambulance as an EMT. While studying abroad at the University of Cambridge, she hitch-hiked across Europe to raise funds to support educational opportunities for children in Africa. To continue her interest in science with medical applications, she decided to pursue graduate studies. During Cornell's graduate school visitation weekend, Debra met Professor Lara Estroff whose passion for Biomineralization and fascinating research got her to sign up. Debra completed her BS in Course 3 at MIT in 2007.
Hydroxyapatite (HAP, Ca 10 (PO 4 ) 6 (OH) 2 ) nanoparticles with controlled materials properties ... more Hydroxyapatite (HAP, Ca 10 (PO 4 ) 6 (OH) 2 ) nanoparticles with controlled materials properties have been synthesized through a two-step hydrothermal aging method to investigate fibronectin (Fn) adsorption. Two distinct populations of HAP nanoparticles have been generated: HAP1 particles had smaller size, plate-like shape, lower crystallinity, and more negative ζ potential than HAP2 particles. We then developed two-dimensional platforms containing HAP and Fn and analyzed both the amount and the conformation of Fn via Forster resonance energy transfer (FRET) at various HAP concentrations. Our FRET analysis reveals that larger amounts of more compact Fn molecules were adsorbed onto HAP1 than onto HAP2 particles. Additionally, our data show that the amount of compact Fn adsorbed increased with increasing HAP concentration due to the formation of nanoparticle agglomerates. We propose that both the surface chemistry of single nanoparticles and the size and morphology of HAP agglomerates play significant roles in the interaction of Fn with HAP. Collectively, our findings suggest that the HAP-induced conformational changes of Fn, a critical mechanotransducer protein involved in the communication of cells with their environment, will ultimately affect downstream cellular behaviors. These results have important implications for our understanding of organic−inorganic interactions in physiological and pathological biomineralization processes such as HAP-related inflammation.
Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by pro... more Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.
The development of hard tissues such as bone and teeth are examples of biomineralization processe... more The development of hard tissues such as bone and teeth are examples of biomineralization processes in which organisms actively direct mineralization. The formation of hard tissues is a highly regulated process involving the coordination of extracellular matrix production by the cells and the mineralization of the matrix network regulated by proteins secreted by the cells. The formation of these tissues follows a sequence of events in a spatial and temporal manner dictated by cellular processes. In addition to the cells involved in the formation and remodeling of hard tissues, such as osteoblasts (OBs) and osteoclasts (OCs) in bone, other cell-types, such as breast and prostate tumor cells, can interact with bone. Two strategies were taken in this thesis to understand how cells are involved in the mineralization process. Cell culture systems were designed to examine cell-mineral interactions or to examine cell-mediated mineral formation. Chapter 1 discusses the requirements for sustaining cell viability and function as well as the various components involved in designing cell culture systems such as cell source, culture method, and mineral source. Culture systems to study cell-mineral interactions require an environment containing the mineral of interest for cells to be cultured upon. Hydroxyapatite (Ca 10 (PO 4) 6 (OH) 2 , HA) is the main mineral component of bone and has been noted to change in size, shape, and crystallinity with age, anatomical location, and disease progression. Chapter 2 describes a two-step method for synthesizing hydroxyapatite nanoparticles with controlled shape, size, and crystallinity in order to evaluate how differences in HA crystal properties can affect cellular response. Three culture systems were developed and described in this work. Two systems were developed to utilize the synthetic hydroxyapatite nanoparticles (prepared in Chap. 2) for examining cell-mineral interactions. In Chapter 3, dry-annealed synthetic and commercial HA particles were combined with polylactide(co-glycolide) (PLG) and spin-coated onto glass substrates to form a film of PLG-HA to examine how changes in crystallinity can affect OB attachment and matrix production. Chapter 4 describes the integration of a series of hydrothermally aged HA nanoparticles into porous PLG scaffolds to examine breast cancer cell response to various HA nanoparticles with discrete sizes. Finally, Chapter 5 details the third system designed for culturing cells in a 3-D gel system that allows for the presence of 1-D gradients to study cell-mediated mineral formation with articular chondrocytes. Due to the breadth of components involved, designing culture systems for mineralization studies are complex and selection of cell source, culture method, and mineral source should be dictated by research questions of interest of particular aspects of these processes (Chapter 1). Chapter 2 demonstrates how controlling for reactant salts, reactant concentration, reaction duration, and reaction volume in the initial precipitation step leads to changes in shape (e.g., rounded to plate-like) and phase (e.g., brushite to HA) while further post-processing techniques can be applied to tune phase, size, crystallinity, and dispersity. Surfaces containing dry-annealed particles increased fibronectin secretion from osteoblasts in Chapter 3, however this response was not due solely to an increase in crystallinity, and other changes to the surfaces due to heat treatment need to be examined. Breast tumor cells in Chapter 4 attached better on scaffolds containing smaller, less crystalline HA, however interleukin-8 (IL-8) secretion was upregulated in cells cultured on scaffolds containing larger, more crystalline HA, indicating IL-8 as a potential factor in facilitating breast cancer tumor metastasis processes. Chapter 5 establishes a 3-D culture system which can support cell viability and matrix production with future work to optimize the system for culturing cells under mineralizing gradients. iii BIOGRAPHICAL SKETCH Debra DengWen Lin was born on September 4, 1985 to TsanHai Lin and ShuFen Huang. Her parents had immigrated to the United States in search of better opportunities and encouraged Debra to do the same. She found science with medical applications to be fascinating and sought colleges with research opportunities in the field. Debra graduated from Auburn High School ranked number 1, varsity tennis MVP, gold medalist pianist, with a bunch of medals from Science Olympiad, and went on to attend the Massachusetts Institute of Technology fall of 2003. At MIT, Debra joined Course 3, the Department of Materials Science & Engineering. During that time, she authored a patent at her summer internship at Toyota Motor Corporation in Japan, pulled an all-nighter with a group of friends to write a successful SBIR grant, and drove around Boston on the MIT ambulance as an EMT. While studying abroad at the University of Cambridge, she hitch-hiked across Europe to raise funds to support educational opportunities for children in Africa. To continue her interest in science with medical applications, she decided to pursue graduate studies. During Cornell's graduate school visitation weekend, Debra met Professor Lara Estroff whose passion for Biomineralization and fascinating research got her to sign up. Debra completed her BS in Course 3 at MIT in 2007.
Hydroxyapatite (HAP, Ca 10 (PO 4 ) 6 (OH) 2 ) nanoparticles with controlled materials properties ... more Hydroxyapatite (HAP, Ca 10 (PO 4 ) 6 (OH) 2 ) nanoparticles with controlled materials properties have been synthesized through a two-step hydrothermal aging method to investigate fibronectin (Fn) adsorption. Two distinct populations of HAP nanoparticles have been generated: HAP1 particles had smaller size, plate-like shape, lower crystallinity, and more negative ζ potential than HAP2 particles. We then developed two-dimensional platforms containing HAP and Fn and analyzed both the amount and the conformation of Fn via Forster resonance energy transfer (FRET) at various HAP concentrations. Our FRET analysis reveals that larger amounts of more compact Fn molecules were adsorbed onto HAP1 than onto HAP2 particles. Additionally, our data show that the amount of compact Fn adsorbed increased with increasing HAP concentration due to the formation of nanoparticle agglomerates. We propose that both the surface chemistry of single nanoparticles and the size and morphology of HAP agglomerates play significant roles in the interaction of Fn with HAP. Collectively, our findings suggest that the HAP-induced conformational changes of Fn, a critical mechanotransducer protein involved in the communication of cells with their environment, will ultimately affect downstream cellular behaviors. These results have important implications for our understanding of organic−inorganic interactions in physiological and pathological biomineralization processes such as HAP-related inflammation.
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