Articular cartilage is a highly specialized connective tissue in the body responsible for protecting and cushioning bony ends in diarthrodial joints. Despite the unique ability of this tough, spongy matrix to absorb repetitive stress and loading, cartilage damage is a common occurrence, and as cartilage possesses poor self-repair capabilities, tissue-engineered cartilage replacement is under development as a viable method of repair. Tissue-engineered constructs have thus far been unable to replicate the matrix composition of native cartilage satisfactorily enough to produce usable mechanical properties; in particular, collagen content is very low. One means of improving engineered construct composition may be pulsed low-intensity ultrasound (PLIUS), which is used clinically to stimulate healing of chronic bone lesions, and has been shown to affect chondrocytes in cartilage explants and engineered constructs. We believe it may be of use specifically in improving collagen quantity and quality in engineered constructs. FT-IR spectroscopy shows promise as a valuable tool in collagen crosslink maturity analysis, replacing the current expensive, complicated standard of HPLC and allowing for high-resolution spatial mapping of components. A spectral parameter has been established in literature as being related to collagen maturity in bone, which we explore as a potential means of assessing collagen quality in our engineered cartilage. The specific aims of this research are twofold: first, to assess whether PLIUS improves primary bovine chondrocyte-seeded poly-glycolic acid (PGA) mesh scaffold composition by culturing groups with and without PLIUS stimulation, and second, to correlate FT-IR parameters (including the aforementioned maturity parameter) from engineered cartilage specimens and pure crosslink peptides to mechanical testing in unconfined compression. / Bioengineering

Activation of platelets is essential for hemostasis. Following damage to the vascular endothelium collagen is exposed, to which platelets stably adhere. After adhesion on collagen, a signaling cascade is initiated, mediated by Glycoprotein VI (GPVI), which results in platelet activation. A major signaling protein in GPVI signaling is Spleen tyrosine kinase (Syk). It undergoes phosphorylation and activation following GPVI stimulation. Syk's central role in this physiological process suggests regulation of its activity is required to maintain the platelets response to collagen within physiological limits. The regulation of Syk activation is the focus of this work. Previously published reports implicate the phosphatases SHP-1, SHP-2 and TULA-2 in the negative regulation of Syk. Therefore, we tested these phosphatases possible role in platelets. We show that SHP-1 can dephosphorylate Syk in vitro, but is unable to bind Syk. Also, Syk is hypophosphorylated in GPVI-stimulated SHP-1 deficient platelets and platelet functional responses are minimally affected compared to wild-type platelets. SHP-2 is unable to bind Syk and Syk is not a good substrate for SHP-2 in vitro. TULA-2 dephosphorylated Syk in vitro and associated with Syk in platelets. In TULA-2 deficient platelets, Syk and PLCγ2 were hyperphosphorylated compared to wild-type platelets. Deletion of TULA-2 resulted in enhanced GPVI-dependent platelet functional responses and a prothrombotic phenotype. c-Cbl has been shown to be a negative regulator of GPVI signaling, possibly by regulating Syk phosphorylation. Thus, SHP-1, SHP-2 and TULA-2’s role in c-Cbl regulation of GPVI was also investigated. We show that TULA-2 is able to bind c-Cbl in platelets. SHP-1 and SHP-2 do not. Furthermore, we show a striking similarity between the phenotype of TULA-2 and c-Cbl deficient platelets. However, in vitro binding studies show TULA-2 is able to bind Syk independently of c-Cbl. Thus, the exact role of c-Cbl in regulating Syk dephosphorylation is unclear. In conclusion, we show SHP-1 and SHP-2 are probably not involved in the negative regulation of Syk. However, TULA-2 is the major phosphatase responsible for the negative regulation of Syk in GPVI signaling. This serves to negatively regulate GPVI-mediated platelet function and prevent uncontrolled platelet activation that could lead to thrombosis. / Pharmacology

The Drosophila heart is a tubular vessel surrounded by a dynamic scaffold of extracellular matrix (ECM) proteins. Heart development and function rely upon protease-mediated remodelling and turnover of the ECM, and changes in ECM composition correlate with age and cardiac disease. Previous research has shown that a family of proteases called matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), are necessary for normal cardiac cell migration and lumenogenesis. The Drosophila heart expands considerably throughout growth, but the role of MMP activity has not been elucidated at this time. I examine the role of the two Drosophila MMPs, MMP1 and MMP2, as well as TIMP, in defining larval heart structure and ECM protein distribution. I observe heart phenotypes via immunofluorescence labelling and confocal microscopy using loss-of-function mutants, gene over-expression, and gene knock-down techniques. Reduced MMP1 function during embryogenesis correlates with myofibrillar disorganisation, whereas reduced MMP2 function or TIMP over-expression both result in cardia bifida as well as increased density and ectopic localisation of Collagen-IV and Pericardin. Post-embryonic MMP reduction compromises cardiac structural integrity but does not affect Pericardin localisation. Live imaging of the larval heart with optical coherence tomography (OCT) and light microscopy reveals that reduced MMP2 function correlates with decreased heart rate but not impaired dilation or contraction. These data suggest that MMP2 activity during embryogenesis is critical for larval heart development. In contrast, post-embryonic protease function appears to have a less pronounced effect on ECM protein distribution throughout larval development. / Thesis / Master of Science (MS) / The fruit fly (Drosophila) heart undergoes significant changes in organisation and size throughout development and growth. The heart is surrounded and supported by a network of extracellular matrix (ECM) proteins, which is regulated by proteases, including matrix metalloproteinases (MMPs). Previous research has shown that MMPs are required for normal heart formation. I demonstrate that a reduction in MMP activity during embryonic development results in larval heart defects and an increase in the disorganisation of ECM proteins around the heart, whereas reduction during larval development results in less pronounced protein mislocalisation. These findings are corroborated via over-expression of an MMP inhibitor.

Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a large homopolymeric glycoprotein that is synthesized and stored by platelets and endothelial cells until activation-induced release. MMRN1 is able to support platelet adhesion through mechanisms involving von Willebrand factor (VWF) and glycoprotein (GP)Ibα, and β3 integrins on activated platelets, and it enhances platelet adhesion to fibrillar collagen, potentially by binding to putative MMRN1-specific GPAGPOGPX (where O is hydroxyproline and X is valine or glutamine) motifs in fibrillar collagens. Using mice with and without selective Mmrn1 deficiency, the goals of this thesis were: 1) further characterize the ability of Mmrn1 to enhance platelet adhesion to collagen, 2) explore the role of fluid shear stress in the ability of Mmrn1 to enhance platelet adhesion, and 3) test the specificity of the GPAGPOGPX motif for Mmrn1 and the ability of GPAGPOGPX to support or enhance platelet adhesion. Mmrn1-deficient (Mmrn1-/-) mouse platelets showed impaired aggregate formation on fibrillar collagen surfaces under high (1500 s-1) and low (300 s-1) shear flow compared to wild-type (Mmrn1+/+) mouse platelets, which was due to reduced initial adhesion and a slower rate of platelet accumulation onto collagen surfaces. Similarly, Mmrn1-/- platelets formed smaller aggregates on immobilized recombinant (r)Vwf surfaces compared to wild-type platelets, and Mmrn1-/- platelets had impaired adhesion and aggregate formation on immobilized murine fibrinogen, but not fibrin, when platelets were pre-activated to release Mmrn1. Type I fibrillar collagen was found to contain a variant of the GPAGPOGPX motif (GPAGPOGPI), and GPAGPOGPX motifs supported adhesion of wild-type, but not Mmrn1-/-, platelets. When presented with the VWF-binding GPRGQOGVMGFO motif and the integrin α2β1-binding GFOGER motif present in fibrillar collagens, the GPAGPOGPX motifs synergistically enhanced platelet adhesion. These findings expand upon the known adhesive functions of platelet multimerin 1 and update knowledge of the motifs that support platelet adhesion to fibrillar collagens. / Dissertation / Doctor of Philosophy (Medical Science)

No description available.

Yes / While achieving the spatial organization of cells within 3D assembled nanofiber/cell constructs via nanofiber-enabled cell layering, the small sizes of inter-fiber pores of the electrospun nanofiber mats could significantly limit cell penetration across the layers for rapid formation of an integrated tissue construct. To address this challenge, efforts were made to improve cell-infiltration of electrospun nanofiber mats by modulating the density distribution and spatial organization of the fibers during electrospinning. Collection of collagen-containing electrospun nanofibers (300–600 nm in diameter) onto the surface of a stainless steel metal mesh (1 mm × 1 mm in mesh size) led to the periodic alternation of fiber density from densely packed to loosely arranged distribution within the same mat, in which the densely packed fibers maintained the structural integrity while the region of loose fibers allowed for cell penetration. Along with improved cell infiltration, the distinct fiber organization between dense and loose fiber regions also induced different morphology of fibroblasts (stellate vs. elongated spindle-like). Assembly of cell-seeded nanofiber sheets into 3D constructs with such periodically organized nanofiber mats further demonstrated their advantages in improving cell penetration across layers in comparison to either random or aligned nanofiber mats. Taken together, modulation of nanofiber density to enlarge the pore size is effective to improve cell infiltration through electrospun mats for better tissue formation. / NSF-IIP. Grant Numbers: 1338958, 1346430; NSF-DMR. Grant Number: 1508511; NSF-CBET. Grant Number: 1033742; and NIAMS. Grant Number: 1R21 AR056416

Japanese quail were used as animal models in four experiments to evaluate the effects of supplementing diets with different lipids on bone chemical, mechanical, and histological properties. In Exp. 1, laying hens were fed a basal diet containing either 5% soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO). The addition of SBO in the maternal diet increased the levels of total n-6 fatty acids and arachidonic acid (AA, 20:4n-6) in yolk and tibial bones of newly hatched progeny (P<0.01), whereas the maternal FO diet elevated the concentrations of total n-3 fatty acids, eicosapentaenoic acid (EPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and total saturated acid, but greatly decreased the amount of AA in both egg and progeny tibiae (P<0.01). The maternal HSBO diet resulted in the accumulation of trans-18:1 fatty acid in egg yolks and tibiae at hatch. The addition of FO or HSBO to the maternal diet significantly lowered the ex vivo PGE2 production of tibiae in newly hatched quail compared to those from hens given the SBO or CF diets (P<0.01). In Exp. 2, the addition of different lipids in the maternal diets did not affect growth, tibial length, diameter or collagen content of the progeny. However, supplementing the maternal diet with 5 % FO or HSBO increased the percent bone ash , increased bone pyridinium crosslinks of collagen, enlarged the cartilaginous proliferative and hypertrophied zones, increased diaphyseal cortical thickness of the tibiae in embryos (P<0.05), and subsequently increased tibial shear force, stiffness (P<0.05) and improved cortical thickness, density and trabecular density in early growth and development of progeny compared to those from hens consuming the SBO or CF diets (P<0.05). In Exp. 3, male quail at one month of age were fed a basal diet containing either 5% SBO, HSBO, CF or FO for seven months. Long-term supplementation in the diets of different lipids did not affect body weight, food intake, tibial length or diameter, but the FO group had the highest tibial percent ash, and both FO and HSBO increased tibial mineral content in aged quail compared to those fed the SBO or CF diets (P<0.05). At 8 months of age, quail fed FO had the highest concentrations of (n-3) fatty acids (20:5n-3, 22:5n-3, 22:6n-3) but the lowest amounts of 20:4n-6 in lipids from tibial cortical bone, whereas the SBO and CF diets greatly elevated (n-6) fatty acids and 20:4n-6 levels. The HSBO diet which contains t18:1 fatty acid resulted in t18:1 accumulation in bone. Long-term supplementation with FO or HSBO increased tibial shear force, stiffness and shear stress, as well as improved cortical thickness and density compared with the SBO or CF diets ( P<.05). In Exp. 4, the addition of SBO or CF to the diet for seven months decreased tibial mineral content compared to the FO diet (P<0.05). Quail fed SBO increased collagen concentration in the tibiae (P<0.05), but the level of collagen crosslinks was higher in quail fed FO or HSBO compared to those given the SBO or CF diets (P<0.05). The PGE2 production in bone organ culture and marrow was greatly increased in quail maintained on the SBO or CF diets (P<0.05). PGE2 production in the bone microenvironment was negatively correlated with the tibial percent ash and collagen crosslinks but had a positive correlation with tibial collagen concentration. The results of these studies demonstrate that either supplementing the maternal diets with or long-term exposure to different lipids alters the chemical composition and metabolism of skeletal tissue in both embryos and aged quail. Maternal dietary SBO or CF had an adverse effect on bone growth and development in embryos. Likewise, long-term exposure to SBO or CF diet impaired bone metabolism and remodeling. In contrast, the FO or HSBO diet had beneficial effects on bone modeling in embryos and remodeling in adult quail. / Ph. D.

Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes. / Doctor of Philosophy / Skin wounds frequently result in scars that can range from barely visible to enormous eyesores. Almost everyone will experience at least one skin wound in their lifetime leading to a scar that they wish were less visible, feeding the multi-billion dollar market for anti-scarring agents. However, many of the products on store shelves that claim to reduce scar appearance have not proven those claims. Most of the therapies that do have some degree of scientific evidence to support their claims are difficult to use properly, such as silicone sheeting, and often result in only minor improvements to scar appearance. Alpha Connexin Carboxy-Terminus 1 (αCT1), marketed in clinical trials as Granexin® gel, is a protein-based therapy that works on the cellular level to fundamentally alter the skin's initial reaction to wounding and improving long-term scar appearance. This dissertation explores the link between cellular processes altered by αCT1 and long-term clinical improvements in scar appearance by studying both the extracellular matrix present in the scar in human and animal models and the creation of that extracellular matrix by dermal fibroblasts. In both human and animal models, topical application of αCT1 had no effect on skin surface appearance at early timepoints of 2-6 weeks, correlating with previous research that found scar appearance only improved at 3+ months post-injury. However, deep within the newly constructed tissue of the scar, these studies show the collagen organizational structure of αCT1-treated scars is more similar to unwounded skin and slightly more dense at early timepoints, suggesting αCT1 marginally improved the speed of healing. These findings in humans and animals were also verified in part in cell culture experiments that found dermal fibroblasts increased collagen output in response to αCT1 treatment. A novel wound healing model in the hairless guinea pig, superior at replicating human skin than established models like the rat, is also presented and shown to have effects strongly similar to the human with αCT1 treatment. These results provide a fundamental insight into the mode-of-action by which αCT1 may improve long term scar appearance and identifies early collagen structure as a target for future therapeutics to modify, as well as a new animal model in which to test them.

The anterior cruciate ligament (ACL) contributes to normal knee function, but it is commonly injured and has poor healing capabilities. Of the current treatments available for ACL reconstruction, none replicate the long-term mechanical properties of the ACL. It was hypothesized that tissue-engineered scaffolds comprised of reconstituted type I collagen fibers would have the potential to yield a more suitable treatment for ACL reconstruction. Ultra-violet (UV) radiation and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were investigated as possible crosslinking methods for the scaffolds, and EDC crosslinking was deemed more appropriate given the gains in strength and stiffness afforded to individual collagen fibers. Scaffolds were composed of 54 collagen fibers, which were made using an extrusion process, organized in accordance with a braid-twist design; the addition of a hydrogel (gelatin) to this scaffold was also investigated. The scaffolds were tested mechanically to determine ultimate tensile strength (UTS), Young's modulus, and viscoelastic properties. Scaffolds were also evaluated for the cellular activity of primary rat lateral collateral ligament (LCL) and medial collateral ligament (MCL) fibroblast cells after 7, 14, and 21 days. The crosslinked scaffolds without gelatin exhibited mechanical and viscoelastic properties that were more similar to the human ACL. Cellular activity on the crosslinked scaffolds without gelatin was observed after 7 and 21 days, but no significant increase was observed with time. Although more studies are needed, these results indicate that a braid- twist scaffold (composed of collagen fibers) has the potential to serve as a scaffold for ACL replacement. / Master of Science

The detection of dairying is essential to understand prehistoric economies, particularly in northwest Europe, where a high degree of lactose tolerance implies that fresh milk has long been a significant dietary component. Domestic cattle (Bos taurus) are biologically able to breed year-round, potentially enabling farmers to select a calving strategy to suit their economic focus. Published literature and interviews with farmers suggests that spring calving would have been favoured by economies focussed on meat or storable dairy products, whereas the year-round provision of fresh milk would have required two calving seasons, in spring and autumn, or an extended period through spring, summer and autumn. This thesis uses intra-tooth isotope ratio analysis of cattle tooth enamel to predict birth seasonality as an indicator of dairying. Analysis was performed on first, second and third cattle molars from the archaeological sites of Mine Howe, Pool and Earl’s Bu (Iron Age and Viking period Orkney), Old Scatness (Iron Age Shetland) and Grimes Graves (Bronze Age Norfolk). Modern molars from Chillingham Wild White cattle were also analysed. A new method to determine cattle birth seasonality has been proposed utilising the isotopic patterning (δ13C, δ18O) recorded within first and second molar enamel. Results suggest that birth seasonality estimates are of sufficient accuracy to discriminate between single-and multiple-season calving. Although Pool and Grimes Graves have been interpreted as dairying sites from their age-at-death slaughter patterns, birth seasonality predictions imply an economy focussed on year-round fresh milk at Pool but an emphasis on storable dairy products at Grimes Graves. In addition, it has been demonstrated that intra-tooth enamel data can provide information regarding sub-annual variation in diet and environment. A new method to investigate weaning strategy has also been proposed.