Browsing by Subject "Extracellular matrix"
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Item A new view of culmination in dictyostelium: morphogenetic roles of cell shapes, intercellular junctions, and extracellular matrices(Texas Tech University, 2000-05) Grimson, Mark JeffreyNot availableItem Extracellular matrix mechanics regulate cell signaling and migratory potential in cancer(2012-05) Srivastava, Jaya, active 2012; Ellington, Andrew D.; Zaman, Muhammad H. (Muhammad Hamid)The objective of the presented research is to examine the relationship between the cellular microenvironment and biochemical response of metastatic cells. Clinically recognized as a trait of cancer progression, the cellular microenvironment can have variable and distinct mechanical properties that are processed via cellular mechanosensing, resulting in a cellular biochemical response. A range of studies investigating the interactions between the cellular micromechanical environment and the cell's molecular response during disease progression have been made, yet remain absent of quantitative characterization of many of these coordinated responses. The fundamental inquiry that drives the following research attempts to elucidate how a cell perceives the physical microenvironment and converts that signal to a biochemical response. With the goal of providing insight to such responses, the presented research seeks to elucidate the following questions: (1) What are the integrated effects of ECM stiffness, ECM architecture, and breast cancer cell metastatic potential on cell migration? (2) How does endogenous tissue transglutaminase (tTG) cross-linking of the ECM scaffold effect ECM mechanical properties? (3) How does the architecture and stiffness of the extracellular matrix (ECM) effect the systems-level cellular migration and signaling response? (4) What are the integrated effects of ECM architecture and the targeted knockdown of integrin [beta]1 and MT1-MMP on cellular metastatic potential? The presented research utilizes an interdisciplinary approach, integrating experimental mechanics, biochemical analysis, cellular biology techniques, covalent chemistry, and various microscopy techniques, to investigate these events. In short, cancerous cells are cultured atop or within synthetic collagen type I ECMs of varying mechanical stiffness and structure. These cells are subsequently analyzed by molecular analysis and immunoassays, including quantitative PCR, Western blotting, and gelatin zymography, to acquire measures of the cellular response to perturbations of micromechanical environment. Time-lapse microscopy experiments and subsequent image analyses enable observations of cellular migratory potential through synthetic ECMs. Results indicate that cooperative synergy between ECM properties, cell-matrix adhesion, and pericellular proteolysis drive cell migratory potential of highly invasive tumorigenic cell populations. Collectively, these findings contribute to the cancer biology and mechanobiology fields by systematically extending current insights of matrix mechanics, cellular signaling, and cellular migratory potential in cancer.Item An immunohistochemical analysis of regenerating cellular material in two distinct models of skeletal muscle injury(2011-08) Sarathy, Apurva; Farrar, Roger P.; Suggs, Laura J.Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct.Item Nanoengineering of surfaces to modulate cell behavior : nanofabrication and the influence of nanopatterned features on the behavior of neurons and preadipocytes(2009-08) Fozdar, David Yash; Chen, ShaochenPromising strategies for treating diseases and conditions like cancer, tissue necrosis from injury, congenital abnormalities, etc., involve replacing pathologic tissue with healthy tissue. Strategies devoted to the development of tissue to restore, maintain, or improve function is called tissue engineering. Engineering tissue requires three components, cells that can proliferate to form tissue, a microenvironment that nourishes the cells, and a tissue scaffold that provides mechanical stability, controls tissue architecture, and aids in mimicking the cell’s natural extracellular matrix (ECM). Currently, there is much focus on designing scaffolds that recapitulate the topology of cells’ ECM, in vivo, which undoubtedly wields structures with nanoscale dimensions. Although it is widely thought that sub-microscale features in the ECM have the greatest vii impact on cell behavior relative to larger structures, interactions between cells and nanostructures surfaces is not well understood. There have been few comprehensive studies elucidating the effects of both feature dimension and geometry on the initial formation and growth of the axons of individual neurons. Reconnecting the axons of neurons in damaged nerves is vital in restoring function. Understanding how neurons react with nanopatterned surfaces will advance development of optimal biomaterials used for reconnecting neural networks Here, we investigated the effects of micro- and nanostructures of various sizes and shape on neurons at the single cell level. Compulsory to studying interactions between cells and sub-cellular structures is having nanofabrication technologies that enable biomaterials to be patterned at the nanoscale. We also present a novel nanofabrication process, coined Flash Imprint Lithography using a Mask Aligner (FILM), used to pattern nanofeatures in UV-curable biomaterials for tissue engineering applications. Using FILM, we were able to pattern 50 nm lines in polyethylene glycol (PEG). We later used FILM to pattern nanowells in PEG to study the effect of the nanowells on the behavior preadipocytes (PAs). Results of our cell experiments with neurons and PAs suggested that incorporating micro- and nanoscale topography on biomaterial surfaces may enhance biomaterials’ ability to constrain cell development. Moreover, we found the FILM process to be a useful fabrication tool for tissue engineering applications.Item Neurotrophic factor combinations and extracellular matrix-based hydrogels for nerve regeneration(2006) Deister, Curt Andrew; Schmidt, Christine E.; Roy, KrishnenduItem Repair of skeletal muscle transection injury with tissue loss(2009-08) Merritt, Edward Kelly, 1979-; Farrar, Roger P.A traumatic skeletal muscle injury that involves the loss of a substantial portion of tissue will not regenerate on its own. Little is understood about the ability of the muscle to recover function after such a defect injury, and few research models exist to further elucidate the repair and regeneration processes of defected skeletal muscle. In the current research, a model of muscle injury was developed in the lateral gastrocnemius (LGAS) of the rat. In this model, the muscle gradually remodels but functional recovery does not occur over 42 days. Repair of the defect with muscle-derived extracellular matrix (ECM), improves the morphology of the LGAS. Blood vessels and myofibers grow into the ECM implant in vivo, but functional recovery does not occur. Addition of bone marrow-derived mesenchymal stem cells (MSCs) to the implanted ECM in the LGAS increases the number of blood vessels and regenerating myofibers within the ECM. Following 42 days of recovery, the cell-seeded ECM implanted LGAS produces significantly higher isometric force than the non-repaired and non-cell seeded ECM muscles. These results suggest that the LGAS muscle defect is a suitable model for the study of traumatic skeletal muscle injury with tissue loss. Additionally, MSCs seeded on an implanted ECM lead to functional restoration of the defected LGAS.Item Resistance training as a modality to enhance muscle regeneration in a rat skeletal muscle defect(2009-12) Taylor, Daniel Ryan; Farrar, Roger P.; Suggs, LauraTraumatic skeletal muscle injuries that include loss of large amounts of muscle mass are becoming more common in today’s warfare. Traditional treatments often do not prevent long term functional impairments. Using a decellularized extracellular matrix (ECM) as scaffolding to replace lost muscle tissue allows for transmission of force through the injury site, and provides a suitable microenvironment receptive to myofiber growth. Seeding the ECM with progenitor cells improves cellular content in the defect area. Exercise exposes the muscle to improved blood flow as well as higher than normal loading. This results in increased blood vessel density as well as higher levels of cellular content, and near complete restoration of function.Item Skeletal muscle repair following Plantar nerve relocation on an extracellular matrix seeded with mesenchymal stem cells in PEGylated fibrin gel as a treatment model for volumetric muscle loss.(2014-08) Da Costa, Adriana Jocelyn; Farrar, Roger P.The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs.Item The impact of mechanical properties of poly(ethylene glycol) hydrogels on vocal fold fibroblasts' behavior(2009-05-15) Liao, HuiminVocal fold scarring, caused by injury and inflammation, presents significant treatment challenges. Tissue engineering might be a promising treatment for vocal fold restoration or regeneration. It is important to investigate how scaffold properties alter cell behavior instead of screening thousand of materials, which is fundamental knowledge for rational scaffold design. This work studies how tuning only one parameter, mechanical strength of the hydrogel scaffold, influences the extracellular matrix production of encapsulated porcine vocal fold fibroblast (PVFF). PVFF cells were encapsulated by photopolymerization in 10 wt%, 20 wt%, and 30 wt% poly(ethylene glycol) diacrylate (PEGDA) hydrogels (MW 10,000), with the similar biochemical environment and network structure but different mechanical properties. Cell adhesive peptide, RGDS, was grafted into each hydrogel network to mimic a cell adhesive environment. The glycosaminoglycans (GAGs) production per cell increased from 10 wt% to 20 wt%, 30 wt% gels, with an increase in hydrogel stiffness. The collagen production per cell increased from 10 wt% to 20 wt% gels but no further increase occurred with the increasing modulus from 20 wt% to 30 wt% gels. Interestingly, in hydrogels of intermediate modulus (20% PEGDA hydrogels), the highest elastin per cell was observed compared with gels with higher and lower storage modulus after day 30. Histological analysis showed GAGs, collagen and elastin were distributed pericellularly. However, the organization of collagen type I appeared to be influenced by gel mechanical properties, which was confirmed by immunohistological analysis. Furthermore, the immunohistological analysis showed that the phenotype of PVFF is regulated by the stiffness of the PEG hydrogel. This study demonstrates that different levels of VFF ECM formation may be achieved by varying the mechanical properties of PEG hydrogels and validates a systematic and controlled platform for further research of cell-biomaterials interaction.