No description available.

No description available.

The current gold standard treatment for bone defects is autologous cancellous bone graft, which involves increased surgery time and donor site morbidity, and limited supply of bone and cells for regeneration. Bioreactors may aid in the generation of mechanically conditioned bone grafts with more cells compared to traditional grafts. However, the specific parameters of fluid flow and mechanical loading which contribute to osteogenesis and cell viability in bioreactors are not fully characterized. Here, a perfusion-compression bioreactor system was developed to study osteogenesis in porcine trabecular bone explants. Loading accuracy was over 88% across six bioreactors at a 0.1 s-1 strain rate and 20 N target force, akin to running. A flow rate of 0.2 mL/min appeared to be more favorable for cell viability than 1 mL/min. Overall, this work offers a foundation for future efforts to enhance cell viability and osteogenesis in bone explants.

The human genome is characterized by an extent of functions that act further than its genetic role. Indeed, the genome can also affect cellular processes by nongenetic means through its physical and structural properties, specifically by exerting mechanical forces that shape nuclear morphology and architecture. The balancing between two chromatin compartments with antagonist functions, namely Transcriptional and Polycomb condensates, is required for preserving nuclear mechanical properties and its perturbation is causative of the pathogenic condition Kabuki syndrome (KS) (Fasciani et al., 2020). KS is a rare monogenic disease caused by the haploinsufficiency in the KMT2D gene encoding for MLL4, a H3K4-specific methyltransferase important for the regulation of gene expression. By interrogating the effect of KMT2D haploinsufficiency in Mesenchymal Stem Cells (MSCs) we discovered that MLL4 loss of function (LoF) impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture and the cell mechanoresponsiveness during differentiation (Fasciani et al., 2020). These results suggest that altered nuclear mechanics rely on chromatin architecture and could potentially lead to changes in cell responses to external mechanical stimuli. In the present work, we investigated the role of Transcriptional and Polycomb condensates in tuning nuclear responses to different external mechano-physical conditions. To affect nuclear mechanics, we employed the use of several mechanical devices (e. g. substrate stiffness, microchannels with constrictions, and cell confinement). We found that Polycomb and Transcriptional condensates are modulated by changes in substrate rigidity in healthy conditions and that MLL4 LoF impairs the MSCs nuclear condensates-driven mechanical response. Furthermore, we observed that MLL4 LoF impacts nuclear adaptation to confined spaces by incrementing susceptibility to nuclear envelope rupture. We also showed that the increased nuclear fragility in MLL4 LoF is accompanied by an alteration of cell migratory capacity and survival rate. Altogether these findings suggest that MLL4 LoF impairs cell responses to external mechanical stimuli, shedding light on the pathological connection between the altered cell mechanoresponsiveness during differentiation and KS phenotype in terms of skeletal and cartilage anomalies.

This study was motivated by a theoretical formulation on mechanobiology of soft and hard skeletal tissue differentiation. To prove this formulation experimentally, I hypothesized that cartilaginous phenotype can be induced in vitro in a seemingly non-cartilaginous cell source from fibrous tissue. In testing this hypothesis, I have focused on cartilage as a target and fibrous tissue as an origin or the source of cell. Four different trials were pursued with one supposition in common, i.e. hydrostatic pressure is one of the main driving forces for chondroinduction in vitro. The first and second trials pertained to the influence of a relatively short and long duration cyclic hydrostatic compression on rat Achilles tendon fibroblasts. The third trial was to examine the effect of two different drugs on cytoskeletal elements of mesenchymal stem cells or mouse embryonic fibroblast lines in pellet cultures combined with the similar duration and/or frequency of cyclic hydrostatic pressure adopted in the aforesaid trials with no pharmacological agents added. Last, attempts were made to implement an advanced technique in molecular biology called 'PCR array' to further quantify expression levels of eighty four pathway-specific genes in mouse TGFbeta/BMP signaling traffic under the same physiological regimen of hydrostatic compression. Results demonstrated that transdifferentation in phenotype from tendon to fibrocartilage may have occurred in vitro in tendon fibroblasts in pellet cultures exposed to hydrostatic pressure. Experiments on the role of the cytoskeleton in mechanotransduction of the applied level of hydrostatic pressure demonstrated that disruption of microfilaments in the presence of cytochalasin-D did not significantly interfere with the anabolic effect of cyclic pressure. However, disruption of microtubule assembly by nocodazole abolished the pressure-induced stimulation in cartilage marker genes. These findings suggest that microtubules, but not microfilaments, are involved in mechanotransduction of hydrostatic pressure by mesenchymal stem cells.

No description available.

No description available.

Metastasis is the leading cause of death among cancer patients. The metastatic cascade, during which cancer cells from the primary tumor reach a distant organ and form multiple secondary tumors, consists of a series of events starting with cancer cells invasion through the surrounding tissue of the primary tumor. Invading cells may perform proteolytic degradation of the surrounding extracellular matrix (ECM) and directed migration in order to disseminate through the tissue. Both of the mentioned processes are profoundly affected by several parameters originating from the tumor microenvironment (extrinsic) and tumor cells themselves (intrinsic). However, due to the complexity of the invasion process and heterogeneity of the tumor tissue, the exact effect of many of these parameters are yet to be elucidated. ECM proteolysis is widely performed by cancer cells to facilitate the invasion process through the dense and highly cross-linked tumor tissue. It has been shown in vivo that the proteolytic activity of the cancer cells correlates with the cross-linking level of their surrounding ECM. Therefore, the first part of this thesis seeks to understand how ECM cross-linking regulates cancer cells proteolytic activity. This chapter first quantitatively characterizes the correlation between ECM cross-linking and the dynamics of cancer cells proteolytic activity and then identifies ß1-integrin subunit as a master regulator of this process. Once cancer cells degrade their immediate ECM, they directionally migrate through it. Bundles of aligned collagen fibers and gradients of soluble growth factors are two well-known cues of directed migration that are abundantly present in tumor tissues stimulating contact guidance and chemotaxis, respectively. While such cues direct the cells towards a specific direction, they are also known to stimulate cell cycle progression. Moreover, due to the complexity of the tumor tissue, cells may be exposed to both cues simultaneously, and this co-stimulation may happen in the same or different directions. Hence, in the next two chapters of this thesis, the effect of cell cycle progression and contact guidance-chemotaxis dual-cue environments on directional migration of invading cells are assessed. First, we show that cell cycle progression affects contact guidance and not random motility of the cells. Next, we show how exposure of cancer cells to contact guidance-chemotaxis dual-cue environments can improve distinctive aspects of cancer invasion depending on the spatial conformation of the two cues. In this dissertation, we strive to achieve the defined milestones by developing novel mathematical and experimental models of cancer invasion as well as utilizing fluorescent time-lapse microscopy and automated image and signal processing techniques. The results of this study improve our knowledge about the role of the studied extrinsic and intrinsic cues in cancer invasion. / Bioengineering / Accompanied by fourteen .avi files.

Migratory cells in vivo move within a predominantly fibrous microenvironment through the action of forces. These dynamic interactions facilitate mechanosensing, critical to fundamental biological processes in pathophysiology. Naturally, the field of mechanobiology has evolved over the past several decades to decipher the role of forces in mechanotransduction using a variety of force-measurement platforms. A central challenge that has yet to be overcome in the field is connecting forces with the interplay between cell shape and ever-changing environment. Here, through design of specific fibrous architectures, a mechanobiological understanding of force feed-forward loop accounting for shape shifting of the environment and cells is developed. Using the non-electrospinning Spinneret Tunable Engineered Parameters (STEP) technique, two complementary force measurement platforms of varying physical attributes are developed to investigate how the force feed-forward loop impacts cell fate. Nanonet Force Microscopy (NFM) comprised of aligned nanonets is designed to study anisotropic cell shapes, while Crosshatch Force Microscopy (CM) comprised of orthogonal arrangement of fibers is designed to study cell bodies of broad shapes. The combination of shapes achieved on these networks recapitulate mesenchymal shapes observed in vivo, which are used to describe cell behaviors not reported before. The new findings include (i) discovery of a new biological structure, termed 3D-perpendicular lateral protrusions (3D-PLPs) which is proposed to be the missing biophysical link in the remodeling of the ECM and perpetuation of desmoplasia. Using NFM, seven discreet steps in formation of force-exerting PLPs anywhere along the cell body is documented, which allow cells to spread laterally and increase in contractility. Using a variety of fiber networks, it is shown that aligned fibers are necessary for PLP formation and suitable environments for myofibroblast activation, and (ii) a force dipole that links matrix deformability with cell contractility. Aided by machine learning, CFM automates the process of fiber feature recognition to measure forces as cells change shapes during migration and differentiate to osteogenic and adipogenic lineages. The force platforms are applied to investigate (i) the bioenergetic contributors fueling cellular migration and a surprisingly overwhelming impact of glycolytic energetic pathway over the traditionally thought mitochondrial energy production is found. However, neither pathway has substantial impact over the cellular force production, and (ii) quantitate the migratory and contractile response of enucleated cytoplasmic fragments naturally shed by cells. A peculiar contractility driven oscillatory migratory phenotype is found, capable of lasting over tens of hours, and absent in intact cells. Overall, new high spatiotemporal capabilities are developed in mechanobiology to quantitate the force-feed forward loops between cell shape and ECM in pathophysiology. / Doctor of Philosophy / Pathophysiology is the study of abnormal changes in the regular body functions of an organism that are causes or consequences of disease onset. Research in this area is mainly focused on identifying the different factors that cause and propagate the disease states such as cancer. Central to many of these processes are events such as cell migration and remodeling of their surrounding environment. The native microenvironment surrounding cells is highly complex and is composed of many classes of macromolecules, with fibrous components being one of the most important. How cells interact with these environments through application of forces and how this further regulates cellular behavior is vital to advancing our understanding of many of these pathophysiological processes. Currently, there is a lack in our understanding of how this dynamic process referred to as the "force feed-forward loop", is perpetuated. This limitation in our understanding can be attributed to the lack of an in vivo mimicking platform that captures this dynamic interaction and is capable of measuring the forces. To this end, the development of two novel single cell force measurement platforms: Nanonet Force Microscopy (NFM) and Crosshatch Force Microscopy (CFM) is presented. These platforms are fiber based systems, generated with the utilization of previously established non-electrospinning technique of Spinneret based Tunable Engineered Parameters (STEP) technique. Using NFM and CFM, forces were computed in wide range of cell shapes from anisotropic to all other spread morphologies. These platforms were applied to identify a new biological structure called perpendicular lateral protrusions and shown to have potential role in the spreading of tumor microenvironment. Furthermore, the force dynamics in physiological processes such as stem cell differentiation into fat cells or bone cells is also identified. How cellular processes such as migration and force production is fueled is also investigated and found to be not heavily reliant on the commonly understood mitochondrial activity. Finally, sub-cellular components known as cell fragments, which are devoid of nucleus, are also observed to be contractile and migratory in nature, independent of parent cell body. These platforms and findings can be further utilized to advance our current knowledge of the progression of these physiological and pathological processes and serve as diagnostic tools for the early identification of disease onset. Furthermore, based on these findings, strategies can be developed for early intervention to inhibit disease progression or devise bioengineered scaffolds for applications in tissue engineering.

It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts are responsible for responding to this tissue damage by breaking down the damaged extracellular matrix (ECM) and secreting a new ECM which aids in wound repair and supports the migration of immune cells. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound repairing fibroblasts. To address this lack of understanding, this thesis focuses on quantifying changes in fibroblast morphology, migratory behavior, and force exertion to investigate this host cell's response to representative cytotoxic (PAO1) and invasive (PA14) strains of P. aeruginosa. These assays study host cell-pathogen interactions on highly aligned nanofibers of varied spacing and diameter, which mimic the fibroblast deposited ECM and dictate fibroblast morphology. We discovered that the cytotoxic strain of P. aeruginosa induced significantly shorter fibroblast death times. Furthermore, two modes of death, sharp and gradual, were identified and found to be dependent on both fiber configuration and strain of P. aeruginosa. In addition, fibroblasts exposed to PAO1 migrating on the parallel formation were found to be significantly slower and less persistent than those exposed to PA14, however, fibroblasts exposed to both strains of bacteria were shown to exert similar forces. Lastly, exposure to PA14 led to the greatest change in actin, evident by increased actin punctae and less prominent actin stress fiber formation. / Master of Science / It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts respond to burn wounds by breaking down the damaged tissue fibers, termed extracellular matrix (ECM), and secreting a new ECM. Unfortunately, severe thermal injuries place hospitalized burn victims at high risk of infection. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound healing fibroblasts. To address this knowledge gap, this thesis focuses on quantifying changes in fibroblast shape, migratory behavior, and force exertion to investigate this host cell's response to two strains of P. aeruginosa, which employ different mechanisms of invasion. These interactions are studied on a platform of suspended nanofibers with controlled spacing and diameter, to dictate fibroblast shape and mimic the fibroblast deposited ECM. We discovered that the two strain of P. aeruginosa induced significantly different fibroblast death times. During death, it was observed that fibroblasts either balled up quickly, termed sharp death, or remained spread out, termed gradual death, dependent upon fibroblast shape and strain of P. aeruginosa introduced. In addition, significant differences in migration speed and persistence were found between fibroblasts exposed to the two strains of bacteria, however, both groups were shown to exert similar forces. Lastly, the fibrous proteins which make up the cytoskeleton of the cell, actin stress fibers, were found to vary among the control and bacteria treated cells.