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In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.

Voice disorders affect up to 9% of the population, and can be caused by vocal fold scarring. They can reduce the ability of a person to participate in the workplace and can also cause depression in individuals. Vocal fold scarring changes the organization and composition of the lamina propria, and affects the biomechanical properties of the tissue. These changes cause an inability of the lamina propria to produce a normal mucosal wave during speech, resulting in hoarseness or complete voice loss. Currently there is a clinical need for treatment options for vocal fold scarring. This work focuses on developing a novel, elastomeric electrospun biomaterial to be used as a model system to evaluate the response of immortalized human vocal fold fibroblast cells (HVFF) to scaffold architecture and to the presence of an elastin polypeptide. The scaffold was developed by electrospinning Tecoflex™. Electrospun scaffolds were successfully made with aligned and unaligned fibers, and they were characterized using scanning electron microscopy (SEM) and uniaxial tensile testing. The aligned scaffolds had initial elastic moduli of ~14 MPa and ~0.3 MPa in the preferred and cross-preferred direction respectively. The unaligned scaffolds had initial elastic moduli of ~5 MPa and ~0.6 MPa in the preferred and cross-preferred direction respectively. An elastin-like polypeptide (ELP) developed in the Woodhouse lab, ELP4, was successfully adsorbed onto the scaffolds to investigate the effect of ELP’s on the HVFF cells. HVFF cells were seeded onto the scaffolds and their viability, proliferation, morphology, and gene expression were characterized. Over the culture period the cells remained viable, and showed signs of proliferation. The scaffold topography had a significant impact on the orientation of the cells, with very aligned cultures on the aligned scaffold, and randomly oriented cells on the unaligned scaffold. The scaffold alignment and the ELP4 coating impacted the extracellular matrix gene expression. The ELP4 coating, and the aligned scaffolds promoted elastin synthesis when tested on day 7, and may also reduce collagen-3 expression on day 3. These results signify that aligned electrospun scaffolds, as well as an ELP4 coating, may be promising to use in future biodegradable vocal fold constructs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-26 09:49:22.891

The treatment of bone defects is facing the situation of lacking donations for autotransplantation. As a valid approach, scaffold-based tissue engineering combines the construction of well-defined porous scaffolds with advanced cell culturing technology to guide tissue regeneration. The role for the scaffold is to provide a suitable environment with a sufficient mechanical stiffness, supports for cell attachment, migration, nutrients and metabolite transport and space for cell remodeling and tissue regeneration. The random copolymers poly(L-lactide-co-ɛ-caprolactone) (poly(LLA-co-CL)) and poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) have been successfully incorporated into 3D porous scaffolds to induce specific interactions with cells and direct osteogenic cell differentiation. In this thesis, these scaffolds have been modified in chemical and physical ways to map and understand requirements for bone regeneration. Scaffold functionalities and properties, such as hydrophilicity, stiffness, size/shape, and reproducibility, were studied. The hydrophilicity was varied by adding 3–20 % (w/w) Tween 80 to poly(LLA-co-CL) and poly(LLA-co-DXO) respectively, which resulted in contact angles from 35° to 15°. With 3 % Tween 80, the resultant mechanical and thermal properties were similar to pristine polymer materials. Tween 80 did not significantly influence cell attachment or proliferation but did stimulate the mRNA expression of osteogenetic markers. The surface functionality and mechanical properties were altered by introducing nanodiamond particles (n-DP) into poly(LLA-co-CL) scaffolds by means of surface physisorption or hybrid blending. Scaffold with n-DP physisorbed showed improved cell attachment, differentiation, and bone reformation. Hybrid n-DP/poly(LLA-co-CL) composites were obtained by direct blending of polylactide modified n-DP (n-DP-PLA) with poly(LLA-coCL). The n-DP-PLA was prepared by sodium hydride-mediated anionic polymerization using n-DP as the initiator. Prepared n-DP-PLA could be dispersed homogenously in organic solvents and blended with poly(LLA-coCL) solution. The n-DP-PLA particles were homogenously distributed in the composite material, which significantly improved mechanical properties. For comparison, the addition of benzoquinone-modified n-DP (n-DP-BQ) did not reinforce poly(LLA-co-CL). This indicated the importance of specific surface grafting, which determined different particle-polymer interactions. For the treatment of critical size defects, a large porous poly(LLA-co-CL) scaffold (12.5 mm diameter × 25 mm thickness) was developed and produced by molding and salt-leaching methods. The large porous scaffolds were evaluated in a scaffold-customized perfusion-based bioreactor system. It was obvious that the scaffold could support improved cell distribution and support the stimulation of human mesenchymal stem cell (hMSC) especially with dynamic flow in a bioreactor. To improve the scaffolding technique, a three-dimensional fiber deposition (3DF) technique was employed to build layer-based scaffolds. Poly(LLA-coCL) scaffolds produced by the 3DF method showed enhanced mechanical properties and a homogeneous distribution of human osteoblasts (hOBs) in the scaffolds. Although poly(LLA-co-CL) was thermally degraded, the degradation did not influence the scaffold mechanical properties. Based on the computerized design, a 3DF scaffold of amorphous copolymer poly(LLAco-CL) provides high-precision control and reproducibility. In summary, the design of porous scaffolds is one of the essential factors in tissue engineering as to mimicking the intrinsic extracellular environment. For bone tissue engineering, an optimized scaffold can maintain a contact angle greater than 35 degrees. Pristine or modified n-DP, introduced as an additive by surface physisorption or direct blending, can improve scaffold mechanical properties and cell response. Various sizes of scaffolds can be easily produced by a mold-mediated salt-leaching method. However, when 100 % reproducibility is required, the 3DF method can be used to create customizable scaffolds. / <p>QC 20140929</p>

We observed that platelets, which mediate thrombus formation, also enhance fibrinolysis by single-chain urokinase (scuPA).  Preliminary data suggested that this enhancement was due to platelet thrombospondin (TSP), which, depending upon the specifics of the environment, changed conformation, influencing its role in the fibrinolytic system.  Results showed that the activity of scuPA was enhanced by platelets, regardless of platelet treatment or protein release, and that TSP could not explain the platelet effect.  Investigation of the underlying mechanism, using the non-cleavable mutant scuPA (K158E) and protease inhibitors, showed that the platelet-dependent enhancement of scuPA arose from the activation to uPA by a serine protease.  Factor VII activating protease (FSAP) was not the protease responsible, since inhibition of platelet FSAP with function-blocking antibodies did not inhibit the platelet effect.  Comparison of plasminogen and plasma kallikrein, using an array of inhibitors, showed that both candidates mirrored the platelet effect.  Further results, including those from assessment of protease activity on platelets against chromogenic substrates and from the evaluation of uPA formation over time, were consistent with the involvement of plasminogen.  In addition, experiments with platelets from plasminogen-deficient mice showed that platelets lacking plasminogen no longer activated scuPA.  The enhancement of scuPA was found to be platelet-dependent, even in plasma; scuPA activation was more efficient when plasminogen was associated with the platelet membrane, compared to in solution; and the presence of membranes was essential to induce rapid lysis by scuPA.  Our findings indicate that platelets stimulate fibrinolysis by scuPA via local activation to uPA by platelet-associated plasminogen, which is activated by scuPA, consistent with a system of reciprocal activation.

Coronary heart disease is a leading cause of death among Americans for which coronary artery bypass graft (CABG) surgery is a standard surgical treatment. The success of CABG surgery is impaired by the compliance mismatch between vascular grafts and native vessels. Tissue engineered vascular grafts (TEVGs) have the potential to be compliance matched and thereby reduce the risk of graft failure. Glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen constructs were fabricated and mechanically tested in a previous study by our research group at 2, 8, and 24 hours of GLUT vapor exposure. Constructs electrospun with tropoelastin in addition to gelatin and fibrinogen fibers were also fabricated and tested for the same amounts of GLUT vapor exposure. The current study details a computational method that was developed to predict the material properties of our constructs for crosslinking times between 2 and 24 hours by interpolation and regression of the 2, 8, and 24 hour crosslinking time data. Matlab and Abaqus were used to determine the optimal combination of fabrication parameters to produce compliance matched constructs. The validity of the method was first tested on a 16 hour crosslinked gelatin/fibrinogen construct of 130μm thickness. The predicted compliance was 0.00059 mmHg-1 while the experimentally determined compliance was 0.00065 mmHg-1, a relative difference of 9.2%. Prior data in our laboratory has shown the compliance of the left anterior descending porcine coronary (LADC) artery to be 0.00071 ± 0.0003 mmHg-1. The optimization algorithm predicts that a 258μm thick construct that is GLUT vapor crosslinked for 8.1 hours would match LADC compliance. The algorithm was expanded to predict the compliance of constructs consisting of alternating layers of tropoelastin/gelatin/fibrinogen and gelatin/fibrinogen. A four layered graft was designed and fabricated using this optimization routine. The layered construct was found to have a compliance of 0.00051 mmHg-1 while the predicted compliance was 0.00061 mmHg-1, a difference of 16%. This is a promising method for matching the compliance of our TEVGs with the native tissue of various specimens.

Insulin-like growth factor 1 (IGF-1) acts as a mediator for several hormones in bone. In rat the IGF-1 gene is processed into distinct mRNA molecules which differ in their 5' and 3' ends. Two major classes of mRNA transcript are produced which contain either exon 1 (Class 1) or exon 2 (Class 2). The main aims of this study were to identify the IGF-1 splice variants produced in bone and investigate possible effects of GH on transcript expression. IGF-1 transcripts were analysed in bone using reverse transcription polymerase chain reaction (RT-PCR) using forward specific primers for exon 1 and exon 2 and reverse primers for exon 5 and 6. Analysis of RNA extracted from either whole tibia, growth plate, primary osteoblasts, and clonal cell lines UMR 106 and ROS 17.6 showed distinct patterns of expression. All IGF-1 splice variants were expressed in bone tissue and primary osteoblasts however none of the variants were detected in UMR 106 cell line and only Class 1 Ea was detected in ROS 17.6 cell line. No obvious change in this pattern of expression was observed on GH stimulation of these primary cells and osteosarcoma cell lines. However, GH was found to stimulate osteoblast growth by enhancing proliferation of a subpopulation of cells but GH did not effect alkaline phosphatase production. GH did not increase total IGF-1 mRNA levels in osteoblasts as detected using RNase Protection assay but there was a change in the splicing profile such that Class 2 transcripts were increased by 150% (p=0.05) and Class 1 transcripts decreased by 35% (p=0.02). Furthermore antisense ODNs directed against the common exon 4 ofIGF-1 caused a dramatic increase in osteoblast apoptosis whereas sense and scrambled control ODNs had no effect. Our data show that IGF-1 is an important constitutively expressed factor in osteoblasts and GH may exert its actions by increasing Class 2 transcripts in bone which could have paracrine effects on neighbouring cells.

Obesity is recognized as a risk factor for adverse pregnancy outcomes. Maternal obesity prevalence has increased in parallel with that in the general population and is associated with an increase in morbidity and mortality for both mother and baby. Obese mothers are more likely to develop gestational diabetes, hypertensive disorders including preeclampsia, thromboembolic complications, miscarriage, and have an increased need for induction of labour. Babies born from obese mothers can be abnormally large (macrosomia) or small for gestational age, and have a higher risk of perinatal death and congenital malformation. Pregnancy induces marked and dynamic changes in energy metabolism, however, the direct effects of pregnancy adipose tissue biology in both normal lean and obese women is still largely unknown. The aim of this thesis was to delineate novel mechanisms by which pregnancy affects adipose tissue biology, and thus infer how obesity might adversely affect pregnancy outcomes. We used an animal model of obesity during pregnancy in which mice were given a high fat diet (HF) to make them obese. We identified that pregnancy was associated with an unexpected curtailment of visceral (mesenteric) adipose tissue mass in HF mice and with an attenuation, rather than worsening of the metabolic impairment expected from the combination of excess dietary fat and insulin resistance/glucose intolerance of pregnancy. To determine the underlying molecular mechanism contributing to this phenotype global gene expression microarray with subsequent pathway analysis and qRT-PCR validation was employed within the visceral adipose tissue. In visceral fat of HF pregnant mice, gene pathways for de novo lipogenesis and lipid storage, inflammation, retinol metabolism, insulin like growth factor and estrogenic signaling showed altered regulation. Given the known role of estrogen on adipose tissue and inflammatory cell function, a hypothesis was generated that altered estrogen receptor (ER)α expression/activation/increased estradiol presence within mesenteric fat formed a unifying molecular mechanism underlying the altered adipose biology and relative amelioration of the metabolic phenotype in HF pregnant mice. To test the ER α hypothesis, a female clonal adipocyte cell line, Chub-S7, and primary visceral and subcutaneous adipocytes from pregnant obese and lean patients were treated with the ERα selective agonist, PPT. PPT downregulated mRNA levels of key genes involved in de novo lipogenesis (ME1, FANS and SCD1 Dgat2), consistent with a direct role for ERα activation in curtailment of fat expansion. Although the primary human study lacked sufficient power to adequately address the hypothesis, PPT significantly suppressed SCD1 mRNA levels in visceral adipocytes of lean women. In parallel with the curtailment of mesenteric fat expansion, HF pregnant mice were found to have increased liver weight and liver triglyceride content. However, this “fatty liver” phenotype was not associated with increased mRNA levels of genes involved in hepatic triglyceride uptake or de novo lipogenesis. This increase in liver triglycerides may be due to an excessive influx of fatty acids from mesenteric fat through the portal vein. In conclusion, pregnancy in obese animals is associated with a beneficial curtailment in mesenteric fat expansion, normalization of metabolic disturbances and reduced adipose inflammation. Increased ERα activation within adipocytes may play a critical role in this phenotype.

Cytochrome P450 enzymes are a superfamily of constitutive and inducible haemoproteins, with a central role in the oxidative metabolism of a wide range of compounds. These enzymes have been shown to play important roles both in tumour development and mediation of chemotherapy. Although cytoclirome P450 was first identified in the liver, evidence has accumulated for the presence of cytochrome P450 in extrahepatic tissues including the brain. The presence of the xenobiotic metabolising cytochrome P450 enzymes in the brain is intriguing because of the possible ramifications on function from small changes in P450 levels. o This project has highlighted the presence of several cytochrome P450 enzymes belonging to families 1, 2 and 3 in normal and neoplastic human brain. o In addition, it is the first study to look at cytochrome P450 expression in a number of different human brains using a variety of molecular biology and biochemical techniques. This methodology has provided some indication as to the inter-individual differences in the levels and types of cytochrome P450 enzymes in human brain tissue. o All the P450s examined were identified in specific regions of brain with CYPlAl and CYP2C8 mRNA being the most fi'equently expressed forms. CYP2D6 mRNA was localised primarily to the substantia nigra of the mid brain. The distribution of individual P450s in brain is important in determining the response of the brain to xenobiotics. o This project has also established the presence of several cytochrome P450 enzymes in neoplastic human brain by immunohistochemistry. CYPlAl and CYPIBI were over-expressed in the majority of tumours at high intensity, and almost 50% of tumours exhibited strong CYP3A expression. P450s are important in the aetiology and treatment of various cancers, the over-expression of CYPIBI was demonstrated in the majority of astrocytomas investigated, with no concomitant expression observed in the adjacent normal tissue. Data are emerging that CYPIBI has the capacity to metabolise a variety of putative human carcinogens, including polycyclic aromatic hydrocarbons and heterocyclic amines. In addition, preliminary findings from our group suggest CYPIBI may metabolise key chemotherapeutic drugs. This observations highlights CYPIBI as a possible target for gene therapy and CYPIB1 activated anti-cancer drugs. The major mechanism of induction of members of the CYPl gene family is via the Ah receptor (AhR) complex. To obtain a clearer picture of the mechanisms involved in the regulation of the CYPIBI gene in the brain, five glioma cell lines were investigated, only one of the five (MOG-G-CCM) exhibited constitutive AhR mRNA expression. The lack of the AhR mRNA expression which is the main mechanism of induction of members of the CYPl gene family paralleled the lack of cytochrome P450 gene expression in four out of the five cell lines. A key finding of this study suggests that, although the glioma cell line (MOG-G- CCM) may constitutively express CYPIBI mRNA, the presence of an inducing agent is required for subsequent protein expression. This finding requires further examination to investigate the factors involved in the regulation of the CYPIBI gene. It may be possibly that CYPIBI mRNA is present in normal tissue, and that translation/production of the accompanying protein is usually repressed suggesting that the expression of CYPIBI protein observed in tumour tissue may be due to derepression.

The development of bone regeneration materials to support new bone formation is an active field of research. This report describes the development and characterization of a novel composite scaffold made of a chitosan-alginate co-polymer hydrogel matrix and eggshell (ES) particles. Scaffolds with ES particles or with nanotextured ES (NTES) particles following treatment with phosphoric acid were compared to scaffolds without particles. The scaffolds with particles exhibited a higher porosity and a larger median pore size. Their mechanical strength remained low, but both scaffold types were more resistant to deformation following compression than the scaffolds without particles. The osteogenic potential of the scaffolds was then evaluated with human bone-marrow derived mesenchymal stem cells (MSCs) from four different donors. Results showed that the inclusion of ES or NTES particles significantly increased MSC adherence and viability, as well as alkaline phosphatase activity in the scaffolds. A change of cell morphology and a small, although not statistically significant, increase of osteogenic protein expression (RUNX2 and osteopontin) were also observed at later time points (days 14 and 21). Overall, this research highlights the potential of ES for bone regeneration applications, opening the door for a high-value repurposing of a current industrial waste product.