Understanding the Role of Macrophage Phenotype in Biomaterial-Mediated Tissue Regeneration

The underlying goal of tissue engineering is to functionally repair and regenerate complex tissues and organs. One of the major challenges in engineering viable tissues is forming functional and stable blood vessel networks (angiogenesis) within the tissue, which supply oxygen and nutrients to the cells. Following implantation, these networks must subsequently connect with the body's existing vasculature (anastomosis) for continued survival. Currently, there is no known way to control anastomosis, preventing the translation of many potentially useful biomaterials for tissue engineering applications. Macrophages, the primary cells of the inflammatory response, are major contributors to vascularization and regulate the response to implanted biomaterials; however, macrophages are highly plastic cells that alter their behavior in response to local stimuli, and the contributions of macrophage phenotype to these processes are poorly understood. Therefore, the overarching goals of this work were to (1) understand how regenerative biomaterials modulate macrophage behavior and (2) delineate the impact of changing macrophage phenotype on biomaterial vascularization. First, the in vitro response of primary human macrophages to biomaterials proven to enhance tissue regeneration in animal models was evaluated. Interestingly, biomaterials more successful in promoting tissue repair induced a phenotypic shift in macrophage behavior toward an anti-inflammatory "M2" state. The modulatory effects of these scaffolds were predominantly due to direct cell-scaffold interactions, as only modest changes in macrophage gene expression were observed by soluble factors derived from the scaffolds. Importantly, these findings provide evidence that regenerative biomaterials modulate macrophage behavior. Then, to elucidate the effects of changing macrophage phenotype on biomaterial vascularization, crosstalk between macrophages and vascular endothelial cells (ECs) was assessed via transwell co-culture. Interestingly, the angiogenic behavior of ECs was differentially influenced by macrophage phenotype; specifically, macrophages stimulated toward M1 and M2c activation induced EC up-regulation of genes related to vessel sprouting, while M2a and M2f macrophages altered genes related to vessel branching and extracellular matrix disassembly, respectively. Finally, the functional consequences of changing macrophage phenotype on biomaterial vascularization were ascertained through development of a 3D in vitro model of vascular growth. Self-assembly of ECs and support cells into vascular structures was achieved by co-culture on commercially available Gelfoam(r) scaffolds, to which macrophages were seeded at different stages of vessel development. Consistent with the previous study, M1 and, to a lesser extent, M2, macrophages increased vessel sprouting and the number of connected vessels relative to vascular networks without macrophages. Preliminary studies also demonstrated the potential for temporal control over macrophage activation to enhance vascularization. Collectively, these findings can be used to inform the design of biomaterials that harness the inflammatory response to promote vascularization and improve healing outcomes. This work also has important implications for treating diseases characterized by extensive blood vessel growth, such as cancer and autoimmune conditions, whereby vascularization of the tissue facilitates disease progression.Ph.D., Biomedical Engineering -- Drexel University, 201

Relationship between nanotopographical alignment and stem cell fate with live imaging and shape analysis

The topography of a biomaterial regulates cellular interactions and determine stem cell fate. A complete understanding of how topographical properties affect cell behavior will allow the rational design of material surfaces that elicit specified biological functions once placed in the body. To this end, we fabricate substrates with aligned or randomly organized fibrous nanostructured topographies. Culturing adipose-derived stem cells (ASCs), we explore the dynamic relationship between the alignment of topography, cell shape and cell differentiation to osteogenic and myogenic lineages. We show aligned topographies differentiate cells towards a satellite cell muscle progenitor state - a distinct cell myogenic lineage responsible for postnatal growth and repair of muscle. We analyze cell shape between the different topographies, using fluorescent time-lapse imaging over 21 days. In contrast to previous work, this allows the direct measurement of cell shape at a given time rather than defining the morphology of the underlying topography and neglecting cell shape. We report quantitative metrics of the time-based morphological behaviors of cell shape in response to differing topographies. This analysis offers insights into the relationship between topography, cell shape and cell differentiation. Cells differentiating towards a myogenic fate on aligned topographies adopt a characteristic elongated shape as well as the alignment of cells

Relationship between nanotopographical alignment and stem cell fate with live imaging and shape analysis

The topography of a biomaterial regulates cellular interactions and determine stem cell fate. A complete understanding of how topographical properties affect cell behavior will allow the rational design of material surfaces that elicit specified biological functions once placed in the body. To this end, we fabricate substrates with aligned or randomly organized fibrous nanostructured topographies. Culturing adipose-derived stem cells (ASCs), we explore the dynamic relationship between the alignment of topography, cell shape and cell differentiation to osteogenic and myogenic lineages. We show aligned topographies differentiate cells towards a satellite cell muscle progenitor state - a distinct cell myogenic lineage responsible for postnatal growth and repair of muscle. We analyze cell shape between the different topographies, using fluorescent time-lapse imaging over 21 days. In contrast to previous work, this allows the direct measurement of cell shape at a given time rather than defining the morphology of the underlying topography and neglecting cell shape. We report quantitative metrics of the time-based morphological behaviors of cell shape in response to differing topographies. This analysis offers insights into the relationship between topography, cell shape and cell differentiation. Cells differentiating towards a myogenic fate on aligned topographies adopt a characteristic elongated shape as well as the alignment of cells

CNN based Repeated Cropping for Photo Composition Enhancement

This paper proposes a novel method for aesthetic photo recomposition using a convolutional neural network (CNN).CNN has been showing remarkable performances in various tasks, such as object detection and recognition, and we exploit its usage for photo recomposition. In our framework, CNN is used to iteratively predict cropping directions for a given photo, generating an aesthetically enhanced photo in terms of composition. Experimental results and user study show that the proposed framework can automatically crop a photo to follow specific composition guidelines, such as the rule of thirds and the salient object size.This paper proposes a novel method for aesthetic photo recomposition using a convolutional neural network (CNN).CNN has been showing remarkable performances in various tasks, such as object detection and recognition, and we exploit its usage for photo recomposition. In our framework, CNN is used to iteratively predict cropping directions for a given photo, generating an aesthetically enhanced photo in terms of composition. Experimental results and user study show that the proposed framework can automatically crop a photo to follow specific composition guidelines, such as the rule of thirds and the salient object size.