Study of collagen organization in cell-laden hydrogels and animal tissue samples for effective tissue engineering scaffolds

The interaction of biomaterials with biological systems is a complex process, that is triggered in response to implants and wounds. It is essential to understand the phases of wound healing response, particularly the interactions of immune cells such as macrophages and fibroblasts, with the local extracellular matrix which can influence implant acceptance or the restoration of the damaged wound site. Materials properties such as compressive modulus, surface geometry, functionalization, and topology can be tuned to modulate the inflammatory and fibrotic responses to wounds and implants. Naturally derived materials, such as alginate, are widely used biomaterials owing to their biocompatibility and the diverse crosslinking strategies that can be used for fabrication. Soft alginate gels can be synthesized after methacrylation to be relatively stable under physiological conditions, while retaining pH sensitivity, which can be useful in the treatment of chronic wounds. Studying the collagen response to NIH/3T3 fibroblasts encapsulated in these soft hydrogels can develop wound healing strategies to promote faster wound healing. The transition of collagen organization from aligned to isotropic states in the dually crosslinked stiffer methacrylated alginate (ALGMA) hydrogels shows promise towards the development of topical gels for wound care. Modifying the surface properties using arginine-like derivatives is effective in modulating the fibroblast response to implanted glass beads in SKH1-E mice. Collagen response to modified glass beads using SHG microscopy was evaluated using several factors such as collagen amount, secretion of collagen III, and organization of collagen. The albizziin modification showed both isotropic collagen organization as well similar collagen type III as unwounded skin. Furthermore, statistical analysis uncovered correlations between SHG derived parameters and the materials properties of the chemical modifiers. Collagen type III was correlated with the surface tension of the modifier, and an empirical equation was derived relating materials parameters with the observed collagen measurements. The effectiveness of diverse wound care strategies on shallow and deep wounds on porcine subjects was conducted using SHG microscopy. Treatment duration, as well as scaffold preparation were instrumental in reducing a scarring response and accelerating wound closure rates. By combining the understanding of wound healing in diverse tissue environments, with environmentally responsive wound dressings, it is possible to improve the quality of life for millions of patients across the world

Quantitative Analysis Techniques for Assessing Organelle Organization and Dynamics in Individual Cells

In biomedical optics and microscopy, the organization and morphology of organelles have been widely studied. In spite of novel imaging techniques, there is still a lack of quantitative tools to easily measure cellular characteristics from image data. Previous studies have explored multiple approaches to assess organelle organization and alignment, resulting in complicated and extensive algorithms that are both subject to multiple steps of image processing and influenced by non-cellular artifacts. In this thesis, a technique called the Modified Blanket Method (MBM) is introduced to quantify organelle organization through measurements of fractal dimension (FD) on a pixel-by-pixel basis. With the use of simulated fractal clouds, it is demonstrated that the MBM is capable of accurately and rapidly quantify FD, having a higher sensitivity to a wider range of FD values compared to previous methods. Furthermore, the MBM could differentiate mitochondrial organization of radiation-resistant A549 lung cancer cells at different time points post-radiation. In later experiments, the MBM is combined with similar computational techniques to quantify fiber alignment and nuclear shape through measurements of directional variance (DV) and nuclear aspect ratio (NAR). The simultaneous use of these tools demonstrated that the organization and alignment of mitochondria and actin of NIH 3T3 cells treated with L-buthionine-sulfoximine (BSO) change over time, having different nuclear shapes as well. It is then concluded the this set of computational tools is capable of providing significant cellular data, which could potentially be employed to understand the cellular dynamics of multiple pathological conditions such as diabetes, Alzheimer’s, Leigh’s syndrome, and myopathy, all of which are known to be influenced by dysfunctional organelles

Topography-Mediated Fibroblast Cell Migration Is Influenced by Direction, Wavelength, and Amplitude

Biophysical stimuli including topography play a crucial role in the regulation of cell morphology, adhesion, migration, and cytoskeleton organization and have been known to be important in biomaterials design for tissue engineering. However, little is known about the individual effects of topographic direction, structure repetition, and feature size of the substrate on which wound healing occurs. We report on the design of a topographical gradient with wavelike features that gradually change in wavelength and amplitude, which provides an efficient platform for an in vitro wound healing assay to investigate fibroblast migration. The wound coverage rate was measured on selected areas with wavelength sizes of 2, 5, and 8 mu m in perpendicular and parallel orientations. Furthermore, a method was developed to produce independently controlled wavelength and amplitude and study which parameter has greater influence. Cell movement was guided by topographical properties, with a lower wrinkle wavelength (2 mu m) eliciting the fastest migration speed, and the migration speed increased with decreasing amplitude. However, when the amplitudes were matched, cells migrated faster on a larger wavelength. This study also highlights the sensitivity of fibroblasts to the topographic orientation, with cells moving faster in the parallel direction of the topography. The overall behavior indicated that the wavelength and amplitude both play an important role in directing cell migration. The collective cell migration was found not to be influenced by altered cell proliferation. These findings provide key insights into topography-triggered cell migration and indicate the necessity for better understanding of material-directed wound healing for designing bio-inductive biomaterials

Biaxial stretch effects on fibroblast-mediated remodeling of fibrin gel equivalents

Mechanical loads play a pivotal role in the growth, maintenance, remodeling, and disease onset in connective tissues. Harnessing the relationship between mechanical signals and how cells remodel their surrounding extracellular matrix would provide new insights into the fundamental processes of wound healing and fibrosis and also assist in the creation of custom-tailored tissue equivalents for use in regenerative medicine. In 3D tissue models, uniaxial cyclic stretch has been shown to stimulate the synthesis and crosslinking of collagen while increasing the matrix density, fiber alignment, stiffness, and tensile strength in the direction of principal stretch. Unfortunately, the profound fiber realignment in these systems render it difficult to differentiate between passive effects and cell-mediated remodeling. Further, these previous studies generally focus on a single level of stretch magnitude and duration, and they also investigate matrix remodeling under a homogeneous strain conditions. Therefore, these studies are not sufficient to establish key information regarding stretch-dependent remodeling for use in tissue engineering and also do not simulate the complex mechanical environment of connective tissue. We first developed a novel in vitro model system using equibiaxial stretch on fibrin gels (early models of wound healing) that enabled the isolation of mechanical effects on cell-mediated matrix remodeling. Using this system we demonstrated that in the absence of in-plane alignment, stretch stimulates fibroblasts to produce a stronger tissue by synthesizing collagen and condensing their surrounding matrix. We then developed dose-response curves for multiple aspects of tissue remodeling as a function of stretch magnitude and duration (intermittent versus continuous stretch). Our results indicate that both the magnitude and the duration per day of stretch are important factors in mechanically induced cell activity, as evidenced by dose-dependent responses of several remodeling metrics in response to these two parameters (UTS, matrix stiffness, collagen content, cell number). In addition, we found that cellularity, collagen content, and resistance to tension increased when the tissues were mechanically loaded intermittently as opposed to continuously. Finally, we developed a novel model system that produces non-homogeneous strain distribution, allowing for the simultaneous study of strain gradients, strain anisotropy, and strain magnitude in 2D and 3D. Establishing a system that produces complex strain distributions provides a more accurate model of the mechanical conditions found in connective tissue, and also allows for the investigation of cellular adaptations to a changing mechanical environment

Amoeboid Shape Dynamics on Flat and Topographically Modified Surfaces

I present an analysis of the shape dynamics of the amoeba Dictyostelium discoideum, a model system for the study of cellular migration. To better understand cellular migration in complicated 3-D environments, cell migration was studied on simple 3-D surfaces, such as cliffs and ridges. D. discoideum interact with surfaces without forming mature focal adhesion complexes. The cellular response to the surface topography was characterized by measuring and looking for patterns in cell shape. Dynamic cell shape is a measure of the interaction between the internal biochemical state of a cell and its external environment. For D. discoideum migrating on flat surfaces, waves of high boundary curvature were observed to travel from the cell front to the cell back. Curvature waves are also easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from the coverslip or cells that are extended over the edge of micro-fabricated cliffs. At the leading edge of adhered cells, these curvature waves are associated with protrusive activity, suggesting that protrusive motion can be thought of as a wave-like process. The wave-like character of protrusions provides a plausible mechanism for the ability of cells to swim in viscous fluids and to navigate complex 3-D topography. Patterning of focal adhesion complexes has previously been implicated in contact guidance (polarization or migration parallel to linear topographical structures). However, significant contact guidance is observed in D. discoideum, which lack focal adhesion complexes. Analyzing the migration of cells on nanogratings of ridges spaced various distances apart, ridges spaced about 1.5 micrometers apart were found to guide cells best. Contact guidance was modeled as an interaction between wave-like processes internal to the cell and the periodicity of the nanograting. The observed wavelength and speed of the oscillations that best couple to the surface are consistent with those of protrusive dynamics. Dynamic sensing via actin or protrusive dynamics might then play a role in contact guidance

Combining Smart Material Platforms and New Computational Tools to Investigate Cell Motility Behavior and Control

Cell-extracellular matrix (ECM) interactions play a critical role in regulating important biological phenomena, including morphogenesis, tissue repair, and disease states. In vivo, cells are subjected to various mechanical, chemical, and electrical cues to collectively guide their functionality within a specific microenvironment. To better understand the mechanisms regulating cell adhesive, differentiation, and motility dynamics, researchers have developed in vitro platforms to synthetically mimic native tissue responses. While important information about cell-ECM interactions have been revealed using these systems, a knowledge gap currently exists regarding how cell responses in static environments relate to the dynamic cell-ECM interaction behaviors observed in vivo. Advances at the intersection of materials science, biophysics, and cell biology have recently enabled the production of dynamic ECM mimics where cells can be exposed to controlled mechanical, electrical or chemical cues to directly decouple cell-ECM related behaviors from cell-cell or cell-environmental factors. Utilization of these dynamic synthetic biomaterials will enable discovery of novel mechanisms fundamental in tissue development, homeostasis, repair, and disease. In this dissertation, the primary goal was to evaluate how mechanical changes in the ECM regulate cell motility and polarization responses. This was accomplished through two major aims: 1) by developing a modular image processing tool that could be applied in complex synthetic in vitro microenvironments to asses cell motility dynamics, and 2) to utilize that tool to advance understanding of mechanobiology and mechanotransduction processes associated with development, wound healing, and disease progression. Therefore, the first portion of this thesis (Chapters 2 and 3) dealt with proof of concept for our newly developed automated cell tracking system, termed ACTIVE (automated contour-based tracking for in vitro environments), while the second portion of this thesis (Chapter 4-7) addressed applying this system in multiple experimental designs to synthesize new knowledge regarding cell-ECM or cell-cell interactions. In Chapter 1, we introduced why cell-ECM interactions are essential for in vivo processes and highlighted the current state of the literature. In Chapter 2, we demonstrated that ACTIVE could achieve greater than 95% segmentation accuracy at multiple cell densities, while improving two-body cell-cell interaction error by up to 43%. In Chapter 3 we showed that ACTIVE could be applied to reveal subtle differences in fibroblast motility atop static wrinkled or static non-wrinkled surfaces at multiple cell densities. In Chapters 4 and 5, we characterized fibroblast motility and intracellular reorganization atop a dynamic shape memory polymer biomaterial, focusing on the role of the Rho-mediated pathway in the observed responses. We then utilized ACTIVE to identify differences in subpopulation dynamics of monoculture versus co-culture endothelial and smooth muscle cells (Chapter 6). In Chapter 7, we applied ACTIVE to investigate E. coli biofilm formation atop poly(dimethylsiloxane) surfaces with varying stiffness and line patterns. Finally, we presented a summary and future work in Chapter 8. Collectively, this work highlights the capabilities of the newly developed ACTIVE tracking system and demonstrates how to synthesize new information about mechanobiology and mechanotransduction processes using dynamic biomaterial platforms

Construction of artificial skin tissue with placode-like structures in well-defined patterns using dielectrophoresis

During embryonic development of animal skin tissue, the skin cells form regular patterns of high cell density (placodes) where hair or feathers will be formed. These placodes are thought to be formed by the aggregation of dermal cells into condensates. The aggregation process is thought to be controlled by a reaction-diffusion mechanism of activator and inhibitor molecules, and involve mechanical forces between cells and cells with the matrix. In this project, placode formation in chicken embryonic skin cells was used as a model system for the study of the mechanism by which the placodes are formed. Artificial aggregates of chicken embryonic skin cells were created by suspending them in a 300 mM low conductivity sorbitol solution and attracting them by positive dielectrophoresis to high field regions within microelectrode arrays by applying a 10 - 20 Vpk-pk 1 MHz signal across the microelectrodes. It was demonstrated that using this method aggregates can be produced in a large variety of patterns and that the distance between the aggregates and aggregate size and shape within the pattern can be controlled effectively. Custom-built image analysis tools were developed in LabVIEW to analyze the patterns formed. The formation of aggregates by dielectrophoresis was followed by an immobilization phase of the resulting patterns inside a gel matrix, forming an artificial skin. Nutrients and oxygen were supplied externally. Long-term incubation of the artificial skin shows that embryonic skin cells in the aggregates were viable and showed behavior similar to that of developing embryonic skin, including further aggregation of the cells and the formation of cell condensates. The domain size was shown to have an influence on the condensation process, with cells in small aggregates forming only one condensate near the centre of the aggregate, and several condensates in larger aggregates. Whilst the distribution of cell condensates within the aggregates in round large aggregates is predominantly random, some line formation could be observed in linear aggregations, indicating some self-organization may be occurring

Design of a three-dimensional in vitro model to elucidate the influence of integrin beta 1 and matrix metalloproteinases in breast cancer remodeling of collagen I

Every year there are nearly two million new cases of invasive breast cancer worldwide and over 500,000 deaths, the majority from metastatic sites. While cellular changes during tumorigenesis and progression have been studied, our understanding of extracellular matrix remodeling, at the fiber level, by individual and collective cellular cohorts remains limited. Furthermore, recent studies suggest that there is a correlation between the organization of collagen I fibers perpendicular to the tumor and patient survival. However, the underlying mechanism of this alignment remains unknown. The central hypothesis proposed in this dissertation is that breast cancer tumors reorganize collagen I fibers perpendicular to the tumor surface via integrin β1 and matrix metalloproteinases (MMPs). To investigate this hypothesis, we developed a novel in vitro assay that replicates collagen I fiber alignment previously reported in vivo and a new quantitative collagen I fiber orientation algorithm. Our studies using multicellular aggregates, derived from the triple negative breast cancer cell line MDA-MB-231, embedded into collagen I matrices and confocal reflectance microscopy provide novel insights into how the local microenvironment is affected and into local orientation of the collagen I fibers near the spheroid-collagen I interface. These results agree well with our computational studies. Furthermore, the viability of the algorithm is demonstrated using both in silico and in vitro derived images, and shows that this algorithm is more accurate than similar algorithms previously published. Using the developed in vitro assay and computational algorithm it is also demonstrated that knocking down integrin β1 reduces the amount of collagen I aligned perpendicularly to the tumor surface, while inhibiting MMP activity using the broad spectrum MMP inhibitor GM6001 increases the amount of collagen I aligned perpendicularly to the tumor surface at early time points. The work presented here has implications in three-dimensional multicellular assays, accurate fiber orientation analysis, and understanding the role of integrins in matrix reorganization and cancer cell metastasis.2019-08-09T00:00:00

Microfluidic-based analysis of 3D cell migration under different biophysical and chemical gradients

Several mechanochemical factors are involved in cell migration, fundamental to establish and maintain the proper organization of multicellular organisms. The alteration of migratory patterns of cells could be related to the development of several pathologies. Focusing this work on tissue regeneration, more specifically wound healing, bone regeneration and blood vessel formation, the main aim of this work is to advance in the understanding of how chemical or physical factors present in the cell niche can regulate the cell movement (fibroblasts, osteoblasts and endothelial cells respectively). In an effort to understand what mechanisms are involved, it has been seen that both the extracellular matrix surrounding tissue cells and the biomolecules present in the cellular microenvironment can affect the behavior of cells [1–3]. In turn, interstitial fluid flow, defined as the convective transport of liquids through the extracellular matrix of tissue, is also capable of altering the morphology and cellular movement. Similarly, biomolecules, such as growth factors or drugs, modify the migration pattern. The main mechanisms studied throughout this thesis have been chemotaxis, durotaxis and rheotaxis. The biological processes for which these analyses have been performed were angiogenesis, wound healing and bone regeneration respectively. For the in vitro study of these variables, and making use of novel microfabrication techniques such as microfluidics, new platforms for 3D cell culture have been developed [4,5]. The microfluidic chips used allow replication of the ex vivo tissue microenvironment through the use of hydrogels and the generation of concentration gradients and controlled fluid flows. It should be noted that the versatility of this technology has allowed us to simultaneously study several microenvironmental factors, such as chemical gradients and matrix stiffness applied to fibroblast culture to understand its behavior in the wound area. In addition, these types of systems allow the visualization and/or monitoring of the cellular response in real time, being able to quantify the cellular migration. For the application of fluid flow, a novel system was designed to avoid the rupture of the hydrogels, allowing to obtain a stable interstitial flow inside the chip chamber. Throughout this thesis, it has been seen that there are several factors involved in 3D cell migration. Not only variables such as the chemical gradient (studied in endothelial cells and fibroblasts) or the rigidity of the extracellular matrix (analyzed in fibroblasts and osteoblasts) affect cells [6,7]. The architecture of the matrix, more specifically the disposition of the fibers that conform this matrix, has been identified as playing an important role in cell migration, also altering the morphology of cells, in this case osteoblasts.[1] N. Movilla, C. Borau, C. Valero, J.M. García-Aznar, Degradation of extracellular matrix regulates osteoblast migration : a microfluidic-based study, Bone. 107 (2018) 10–17.[2] O. Moreno-Arotzena, C. Borau, N. Movilla, M. Vicente-Manzanares, J.M. García-Aznar, Fibroblast Migration in 3D is Controlled by Haptotaxis in a Non-muscle Myosin II-Dependent Manner, Ann. Biomed. Eng. (2015). doi:10.1007/s10439-015-1343-2.[3] O. Moreno-Arotzena, G. Mendoza, M. Cóndor, T. Rüberg, J.M. García-Aznar, Inducing chemotactic and haptotactic cues in microfluidic devices for three-dimensional in vitro assays, Biomicrofluidics. 64122 (2014). doi:10.1063/1.4903948.[4] W.A. Farahat, L.B. Wood, I.K. Zervantonakis, A. Schor, S. Ong, D. Neal, R.D. Kamm, H.H. Asada, Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures., PLoS One. 7 (2012) e37333. doi:10.1371/journal.pone.0037333.[5] Y. Shin, S. Han, J.S. Jeon, K. Yamamoto, I.K. Zervantonakis, R. Sudo, R.D. Kamm, S. Chung, Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels, Nat Protoc. 7 (2012) 1247–1259. doi:10.1038/nprot.2012.051.[6] C. Del Amo, C. Borau, R. Gutiérrez, J. Asín, J.M. García-Aznar, Quantification of angiogenic sprouting under different growth factors in a microfluidic platform, J. Biomech. 49 (2016) 1340–1346. doi:10.1016/j.jbiomech.2015.10.026.[7] C. Del Amo, C. Borau, N. Movilla, J. Asín, J.M. Garcia-Aznar, Quantifying 3D chemotaxis in microfluidic-based chips with step gradients of collagen hydrogel concentrations, Integr. Biol. (2017) 1–27. doi:10.1039/C7IB00022G.<br /

Texture based vein biometrics for human identification : A comparative study

Hand vein biometric is an important modality for human authentication and liveness detection in many applications. Reliable feature extraction is vital to any biometric system. Over the past years, two major categories of vein features, namely vein structures and vein image textures, were proposed for hand dorsal vein based biometric identification. Of them, texture features seem important as it can combine skin micro-textures along with vein properties. In this study, we have performed a comparative study to identify potential texture features and feature-classifier combination that produce efficient vein biometric systems. Seven texture features (HOG, GABOR, GLCM, SSF, DWT, WPT, and LBP) and three multiclass classifiers (LDA, ESVM, and KNN) were explored towards the supervised identification of human from vein images. An experiment with 400 infrared (IR) hand images from 40 adults indicates the superior performance of the histogram of oriented gradients (HOG) and simple local statistical feature (SSF) with LDA and ESVM classifiers in terms of average accuracy (> 90%), average Fscore (> 58%) and average specificity (>93%). The decision-level fusion of the LDA and ESVM classifier with single texture features showed improved performances (by 2.2 to 13.2% of average Fscore) over individual classifier for human identification with IR hand vein images.Proceedings - International Computer Software and Applications Conferenc