published_or_final_version / Dentistry / Master / Master of Orthodontics

The notochord is a conserved structure in the phylum chordate, which includes all vertebrates and some closely related invertebrates. In mouse embryos, the notochord is a midline structure underneath the neural tube and it consists of a rod of cells constrained by a thick extracellular sheath, which is rich in fibronectin and other extracellular matrix molecule. During notochord formation, two key processes are involved: cell migration and convergent extension. Two molecules are essential for these processes: fibronectin and Protein Kinase C iota (prkci). For cell migration, fibronectin regulates this process by modulating cell protrusion via a signaling pathway in which atypical protein kinase C (aPKC) is an essential factor. For convergent extension, fibronectin has been shown to be important in this process by regulating both cell migration and cell adhesion. The role of aPKCin convergent extension is revealed as it is asymmetrically expressed in Ciona notochord during convergent extension, indicating possible function of aPKC in convergent extension process and in the morphogenesis of notochord. These studies raised the possibility that fibronectin and prkci are important in notochord formation, by regulating cell migration and convergent extension. In this thesis, Cre/loxP system was used to study the function of fibronectin and prkci in the development of notochord. I provided evidence that conditional deletion of fibronectin by Foxa2-Crein the notochord resulted in a notochord of smaller volume and fewer notochordal cells. The nucleus pulposus was also smaller in are and less in cell number. Fibronectin in the notochord was not affected at E9.5, but diminished in the core of the notochord at E12.5 and in nucleus pulposus at E15.5. The phenotypes of smaller notochord and the nucleus pulposus might be the results of reduced notochordal cell proliferation and increased cell death. However, more samples are needed to analyze to confirm this and perform statistical analysis. In addition, convergent extension of notochord seemed less effective. These results are consistent with previous study about fibronectin and α5β1 integrin. The results suggest that fibronectin is required for notochordal cell proliferation, survival, migration and efficient convergent extension, but not for notochordal cell fate determination. The results also demonstrated that prkci seemed not to be important for notochord development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Endochondral bone development depends on the progression of chondrocyte proliferation, hypertrophy and terminal differentiation, which requires precise transcriptional regulation and signaling coordination. Disturbance of this process would disrupt chondrocyte differentiation and lead to chondrodysplasias. In cells, a highly conserved mechanism, ER stress signaling, has been developed to sense the protein load and maintain the cellular homeostasis. In humans, mutations in COL10A1 induce ER stress and result in metaphyseal chondrodysplasia type Schmid (MCDS). Previous analysis of a MCDS mouse model (13deltg mouse) had revealed a novel mechanism of chondrocyte adaptation to ER stress. The hypertrophic chondrocytes survive ER stress by reverting to a pre-hypertrophic like state (Tsang et al., 2007). To dissect the underlying mechanisms that coordinate chondrocyte survival, reverted differentiation and adaptation to ER stress, different chondrocyte populations in the wild type and 13del growth plates were fractionated for global gene expression analyses. The genome-wide expression profiles of proliferating chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes and terminally differentiated chondrocytes in the wild type growth plate provide molecular bases to understand the processes underlying both physiological and pathological bone growth. Systematic analyses of these transcriptomic data revealed the gene expression patterns and correlation in the dynamics of endochondral ossification. Genes associated with sterol metabolism and cholesterol biosynthesis are enriched in the prehypertrophic chondrocytes. Selected genes (Wwp2, Zbtb20, Ppa1 and Ptgis) that may potentially contribute to endochondral ossification were identified differentially expressed in the growth plate. Bioinformatics approaches were applied to predict regulatory networks in chondrocytes at different differentiation stages, implying the essential and dominant roles of Sox9 in coordination of stage specific gene expression. We further confirmed that Sox9 directly regulates the transcription of Cyr61, Lmo4, Ppa1, Ptch1 and Trps1, suggesting that Sox9 integrates different steps of chondrocyte differentiation via regulation of its target genes and partially crosstalk with IHH signaling pathway. The information on gene expression and regulation from physiological growth plate provides important basis to understand the molecular defects of chondrodysplasia. The hypertrophic zone in 13del growth plate was fractionated into upper, middle and lower parts for microarray profiling, corresponding for the onset of ER stress, onset of reverted differentiation and adaptation phase. Comparative transcriptomics of wild type and 13del growth plates revealed genes related to glucose, amino acid and lipid metabolisms are up regulated in response to ER stress. Fgf21 was identified as a novel ER stress inducible factor regulated by ATF4. Removal of Fgf21 results in increasing cell apoptosis in 13del hypertrophic zone without affecting the reverted differentiation process. Up regulation of genes expression related to hypoxic stress (Slc2a1, Hyou1, Stc2 and Galectin3) in 13del hypertrophic chondrocytes suggested that survival and adaptation of chondrocytes to ER stress involve cross-regulation by other stress pathways. Our findings have provided a new insight into the mechanisms that facilitate chondrocyte survival under ER stress in vivo, and propose the integrative effects of hypoxic stress pathway during the stress adaptation process, which broaden the molecular horizons underlying chondrodysplasias caused by protein folding mutations. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Next Generation Sequencing (NGS) technologies have dramatically increased the throughput and lowered the cost of DNA sequencing. In this thesis, I apply these technologies to unresolved questions in skeletal development and disease. Firstly, I use targeted re-sequencing of genomic DNA to identify the genetic cause of the cartilage tumor syndrome, metachondromatosis (MC). I show that the majority of MC patients carry heterozygous loss-of-function mutations in the PTPN11 gene, which encodes a phosphatase, SHP2, involved in many signaling pathways. Furthermore, I show that cartilage lesions in MC patients likely arise following somatic second-hit mutations in PTPN11. Secondly, I use RNA-seq to identify gene expression changes that occur following genetic inactivation of Ptpn11 in mouse chondrocyte cultures. I show that chondrocytes lacking Ptpn11 fail to properly undergo terminal differentiation and instead continue to express genes associated with earlier stages of chondrocyte maturation. I validate these findings in vivo by examining markers of specific chondrocyte maturation stages in the vertebral growth plates of mice following postnatal mosaic inactivation of Ptpn11. Together, my results provide insight into the molecular mechanisms underlying the initiation and growth of cartilage tumors. In the third component of my thesis, I develop a method to map and clone zebrafish mutations by performing whole genome sequencing on pooled DNA. I apply this method to zebrafish mutants identified in a mutagenesis screen for adult phenotypes, including skeletal phenotypes, and determine that a nonsense mutation in bmp1a underlies the craniofacial phenotype in the wdd mutant. In summary, I show that NGS technologies can be successfully utilized to firstly identify the genetic cause of a human skeletal disorder, secondly investigate the molecular mechanisms regulating the maturation of skeletal cells, and thirdly expedite the process of mapping and cloning zebrafish mutants with skeletal phenotypes. Altogether, my research provides insight into the pathways and processes regulating skeletal development and disease.

Polymer scaffold use has become commonplace in tissue engineering strategies. Scaffolds provide sturdy interfaces that securely anchor tissue engineered constructs to their designated locations. Researchers have used scaffolds to provide support to developing tissues as well as a growth template to aid the development of the desired phenotypic structure. In addition to using scaffolds for their mechanical support, scaffolds can be used as a diagnostic tool by attaching sensors. Strain gauge sensors have been attached to scaffolds to monitor compression and elongation. These polybutylterphalate (PBT) scaffolds were used in a cartilage tissue-engineering project for femoral cartilage repair. The aim of this project was to measure native cartilage pressure in normal canine stifle joints using strain gauge scaffolds. By using pressure sensitive films to confirm joint surface pressures determined with strain gauge measurements, "sensate" scaffolds were created to be able to provide in vivo joint loading measurements. An understanding of the in vivo pressures in the menisco-femoral joint space will facilitate the development of tissue engineered cartilage by determining chondrocyte mechanical triggers as well as helping define reasonable expectations for engineered articular cartilage tissue that is required for successful cartilage repair.

Osteoarthritis is the end result of damage to articular cartilage, which lacks the ability to self-repair. Tissue engineering of cartilage is a promising field of study that aims to promote healing of cartilage in vivo by manipulation of the chondrocytes that maintain the tissue, or through in vitro production of new cartilage for implantation into cartilage defects. Tissue-engineered cartilage constructs require mechanical stimulation to produce matrix components in quantities and proportions similar to native cartilage tissue, and adenosine triphosphate (ATP) is thought to be an autocrine/paracrine biochemical mediator of these mechanical forces on the cell, after its release from chondrocytes under mechanical stress. This study determined culture conditions for chondrocytes in 3D agarose scaffolds from mature donors undergoing total joint arthroplasty for the treatment of osteoarthritis, then supplemented these cells in vitro with exogenous ATP in concentrations varying from 50 nM to 1 mM in the presence of the radioisotopes [35S] and [3H]-proline, with radioisotope incorporation acting as markers of proteoglycan and collagen synthesis respectively. The basal concentrations of ATP in the chondrocyte cultures as well as the ATP half-life in the cultures were determined by lucifer/luciferase assay and luminometry. The P2Y receptor expression on the populations of chondrocytes from 8 donors was determined by flow cytometry, with largely varied individual expression and heterogeneity of P2Y1 and P2Y2 receptors. Exogenous ATP was found to increase synthesis of matrix components by 200% of the control cultures at doses of 100 nM to 1 µM. Patients with worse arthritis patterns, who were on chronic narcotic medications and who smoked were more likely to have a negative response to the exogenous ATP supplementation. The basal concentration of ATP in the cultures was less than 1 nM, and the ATP half-life varied from 1-2 hours, depending on the expression of P2Y1 receptors expressed by the donor’s chondrocyte population (R2 = 0.99). Supplementation of exogenous ATP to tissue-engineered cartilage in vitro appears to be a promising technique for improving the matrix synthesis of these constructs. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2011-01-28 10:49:47.118

Long-term applicability of current surgical interventions for the repair of articular cartilage is jeopardized by the formation of mechanically inferior repair tissue. Cartilage tissue engineering offers the possibility of developing functional repair tissue, similar to that of native cartilage, enabling long-lasting repair of cartilage defects. Current techniques, however, rely on the need for a large number of cells, requiring substantial harvesting of donor tissue or a separate cell expansion phase. As routine cell expansion methods tend to elicit negative effects on cell function, the following study describes an approach to generate large-sized engineered cartilage constructs (≥ 3 cm2) directly from a small number of immature rabbit chondrocytes (approximately 20,000), without the use of a scaffold. After characterizing the hyaline-like engineered constructs, the in vivo repair capacity was assessed in a chondral defect model in the patellar groove of rabbits. In vitro remodeling of the constructs developed in the bioreactor occurred as early as 3 weeks, with the histological staining exhibiting zonal differences throughout the depth of the tissue. With culturing parameters optimized (3 weeks growth under 15 mM NaHCO3), constructs were grown and implanted into critical-sized 4 mm chondral defects. Assessed after 1, 3 and 6 months (n=6), implants were scored macroscopically to evaluate integration and survival of the implants. Out of 18 rabbits, 16 received normal or nearly normal over-all repair assessment. Histological and immunohistochemical evaluation showed good integration with surrounding cartilage and underlying subchondral bone. Architectural remodeling of the constructs was present at each time point, with the presence of flattened chondrocytes at the implant surface and columnar arrangement of chondrocytes in deeper zones. The observation of in vivo remodeling was also supported by the changes in biochemical composition of the constructs. At each time point, constructs had a collagen to proteoglycan ratio similar to that of native cartilage (3:1 collagen to proteoglycan). In contrast, the repair tissue for each control group was inferior to that produced with treated defects. These initial results hold promise for the generation of engineered articular cartilage for the clinical repair of cartilage defects without the limitations of current surgical repair strategies. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-01-31 01:03:15.276

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