Thesis (Ph. D.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

Science and medicine have progressed in unfathomable ways over the past century. Paradoxically, as our result of our advancements in medicine we live in a progressively aging society where the majority of people will have multiple morbidities associated with senescence. The World Health Organization estimates that nearly 100% of the population will suffer dental maladies, which left untreated compound with age. We hope to gain new biomedical insight applicable to the advancing field of dental regenerative therapeutics. This dissertation reveals new dental biology through studying the embryology, genetics and evolution of teeth across patterning, morphogenesis and regeneration. We exploit an innovative model, the Lake Malawi cichlid fishes, to study these processes in a natural system. Malawi cichlids have rapidly evolved diverse species-specific dentitions, ranging from hundreds to thousands of teeth that represent a rainbow of shapes and sizes, yet Malawi cichlid species has nearly identical genomes, offering us a powerful genetic system. Furthermore, unlike classic vertebrate models in embryology such as zebrafish, chicken or mice, cichlids have oral teeth and the ability to replace each tooth constantly throughout their lifetimes. In the first study, we break-down the process of whole de-novo tooth replacement in cichlids. We then explore the re-deployment of initiating gene pathways later in the morphogenesis of each replacement tooth (RT). In the second study we investigate the co-patterning of two placode derived oral organs, teeth and taste buds (TBs), and uncover new genes that may modulate their number and size. We subsequently discover a bipotency of progenitor tissue to form both organs and a later plasticity to trans-fate it through coordination of a Wnt-BMP- Hh genetic hierarchy. In the last study, we explore the stem cells that are responsible for the phenomenon of lifelong cichlid tooth replacement. We describe a common epithelium connected to TBs and rich in stem cells, with a newly discovered stem cell niche at the tip of the RT. We uncover the transcriptomes of both organs, and through differential gene expression informed manipulations, coerce dental cells to display TB characteristics. We hypothesize that TB stem cells may be used in dental therapeutics.

The failure of regeneration of oak (Quercus spp.) and other hardwood species has been noted throughout eastern North America as well as on the eastern Edwards Plateau of central Texas. Previous research has suggested that two factors—prolonged periods of fire suppression and high densities of white-tailed deer—may be particularly influential in preventing seedlings of certain species from reaching the adult size class. It is also possible that these two factors interact, and the success of reintroducing fire to promote hardwood regeneration may depend on local deer density. This study, composed of observational and experimental components, first compared browsing frequency on woody plants in burned and unburned plots at six sites in central Texas. We found that although fire history did not affect browsing frequency, browsing frequency varied significantly among sites (likely due to differences in local deer densities) and among species. In our experimental study, we used cages to protect woody plants from deer herbivory in burned and unburned areas and compared growth after one year to plants of similar size and species that were exposed to herbivory. Plants in burned areas were significantly more likely to increase in height than plants in unburned areas. Similarly, plants protected from herbivory were more likely to increase in height after one year than plants that were browsed. There were no significant differences in the amount of growth woody plants in burned and unburned areas or plants that were caged or uncaged put on during one year. This is likely due to the extreme hot and dry weather that occurred across central Texas during the course of this study. These results support previous research showing that deer are currently limiting hardwood growth of multiple species on the eastern Edwards Plateau. Further, although it appears that fire can be used to stimulate hardwood growth (particularly during a non-drought year) successful hardwood regeneration is unlikely at current deer densities. / text

Skeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury. / text

published_or_final_version / Dental Surgery / Master / Master of Dental Surgery

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

The golden method of peripheral nerve system injury is the nerve autograft, but it is associated with drawbacks such as donor site morbidity, needs of second incisions and the shortage of nerve grafts. Comparatively, connecting the nerve defect directly is an alternative. Unfortunately, if the defects are long, the induced tension will deteriorate the nerve regeneration. These limitations led to the development of artificial nerve guidance conduit (NGC). The market available NGC have problems of unsatisfactory functional recovery and may collapse after the implantation. These are attributed to material and structural deficiencies. Therefore, there is essential to study a biomaterial, which has excellent biological and physical properties to fit the NGC application. In addition, some studies suggested that the poor functional recovery resulted from the NGC implantation were due to the lack of micro-guidance inside the conduit. Thus, it is necessary to investigate the structural influence on the functional recovery of peripheral nerve injury. Crosslinked urethane-doped polyester elastomer (CUPE) is newly invented for a blood vessel graft because it possesses similar mechanical properties of blood vessel which is similar to nerve as well. Therefore, CUPE was also considered to be the NGC. Its biocompatibility has been proved to be excellent in the previous study done by Dr. Andrew SL, Ip. Targeting on the long peripheral nerve regeneration, the aims of this study are (1) to investigate the biocompatibility of CUPE in in-vitro condition and (2) to study the influence of nerve-like structure on the peripheral nerve system injury in an animal model. The ultimate goal is to enhance the functional recovery of peripheral nerve system injury by implanting a flexible biomaterial, CUPE, which has a nerve-like microarchitecture. It is hypothesized that the nerve-like structure can promote the axonal regeneration. The surface energy and roughness of CUPE were investigated. It showed a relatively low surface energy compared to other conventional biopolymers such that the cell adhesion and also the proliferation were inhibited. Therefore, the CUPE was modified by the immersion into a high glucose DMEM. The change in the hydrophilicity, roughness and cell viability of medium treated CUPE were studied. The hydrophilicity of treated CUPE was increased but the roughness was remaining unchanged whereas the pH of the immersion solution did not cause any effect on the cell activity on the CUPE. In the pilot animal study, five channels along the CUPE-NGC had a similar myelinated fiber density and population compared to the nerve autograft. Also, the channels in the CUPE-NGC were fragmented. In summary, the medium treatment could enhance the hydrophilicity of CUPE and the cell activity on CUPE. Such modifications did not governed by the pH of the medium. The NGC-CUPE with five channels, which imitated a basic nerve structure was shown to have a similar tissue regeneration and the functional recovery as the nerve autograft did. The results proved the hypothesis that the nerve-like structure can promote the functional recovery of peripheral nerve system injury with the use of a new biomaterial, CUPE as the NGC substrate. / published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Background Distraction osteogenesis (DO) was first applied on the human craniofacial skeleton in 1992 by McCarthy et al.1 who performed lengthening of the mandible in patients with hemifacial microsomia and Nager’s syndrome. Further advances in this field have since then led to the widespread use of this modality for the treatment of numerous congenital and acquired craniofacial skeletal anomalies. In 2001, a review by Swennen et al2 concluded that up to year 1999, this form of treatment was gaining intense popularity but that the main drawbacks included insufficient data on long term results and relapse. A systematic review of the last decade on functional outcomes and long term complications following distraction osteogenesis of the facial skeleton is presented. Methods A structured systematic literature search, with predefined inclusion and exclusion criteria from relevant computer databases and journals were performed. The journals were evaluated and critically appraised by 2 reviewers separately in 3 rounds. Papers were categorized according to the level of evidence, the quality of methodology and the specific field of functional outcomes and long term complications. Results were then categorized according to the type of distraction movements, ie maxillary advancement and mandibular lengthening. Results A total of 42 papers comprising of 16 studies for maxillary advancement and 26 studies for mandibular lengthening were included in this review. Maxillary advancement was found to be beneficial in patients with cleft maxillary hypoplasia in terms of achieving aesthetic outcome but the risk for velopharyngeal insufficiency remains uncertain. The achieved maxillary advancement was stable if performed on adult patients while a recurrence of midface retrusion was noted if DO was performed on growing patients. Overcorrection was recommended in these cases to an estimated value of 20-50%. Mandibular lengthening was 99% successful in relieving respiratory obstruction in patients with isolated Pierre Robin Sequence (PRS) or syndromic micrognathic infants preventing the need for tracheostomy in the long term, and in 89% successfully decannulating infants with pre-existing tracheostomy. However, feeding and growth outcomes after airway obstruction was relieved remain unknown due to lack of sufficient evidence. Unilateral mandibular DO was successful in achieving aesthetic symmetrical facial balance in patients with hemifacial microsomia however a total loss of corrected distraction length was noted by the end of growth period if DO was performed during growth. Conclusions DO achieved stable results in terms of lengthening the maxilla and mandible but was also noted to cause restricted growth potential of the distracted bone. Hence, the benefits of performing DO during active growth should be weighed against the likely need for a second surgery due to a growth deficit of distracted bone and future surrounding bone growth. However DO in adults remains an alternative to conventional orthognathic surgery and choice of treatment should be patient centred. / published_or_final_version / Dental Surgery / Master / Master of Dental Surgery

Spinal cord injury (SCI) results in extensive demyelination, leading to deleterious axon degeneration and inability of functional recovery. Remyelination has become a part of the fundamental strategy for SCI repair. Endogenous neural progenitor cells (NPCs) respond to SCI producing progenies and provide a possible source of regenerated oligodedrocytes for remyelination. During development of the central nervous system, glycogen synthase kinase-3 isoform beta (GSK-3β) is involved in multiple pathways that regulate oligodendrocyte differentiation and myelination, and thus may also play an important part in remyelination after SCI. This study aims to investigate (1) the role of GSK-3β in the differentiation of adult spinal cord derived-neural progenitor cells (ASC-NPCs); (2) whether AR-A014418 as a GSK-3β inhibitor, can promote oligodendroglial differentiation of ASC-NPCs; (3) the effect of LiCl, another GSK-3β inhibitor, on functional recovery after SCI; (4) the effects of LiCl on the myelin and axonal preservation after SCI. Neurosphere culture from adult mouse spinal cord was performed to test the effect of GSK-3β inhibitors, LiCl and AR-A014418, on differentiation of ASC-NPCs. Phenotyping of differentiated ASC-NPCs by immunocytochemistry (ICC) was performed to identify oligodendroglia progenitor cells (OPCs) at different stages. It was shown that LiCl (1 mM) and AR-A014418 (5 μM) promoted differentiation of OPCs as labeled by oligodendrocyte lineage-specific markers: PDGFR-α, NG2 and O4, while AR-A014418 was more potent in the OPC differentiation. Moreover, preliminary data from western blot confirmed that ARA014418 (5 μM) treatment increased the expression level of pGSK (inactive form of GSK-3) in differentiated ASC-NPCs. This suggests a possible strategy to modulate endogenous NPC response to SCI: to induce the preferential differentiation of NPCs into oligodendrocyte lineage by inhibiting GSK-3β activity and thus leading to enhanced remyelination by the differentiated oligodendrocytes. Basso Mouse Scale (BMS) open field test was used to evaluate the locomotive function of the spinal cord injured mice. The result showed that LiCl (4 mM, 200 μl) administration delivered locally at the lesion site by osmotic pump for 2 weeks improved functional recovery after SCI. Furthermore, immunohistochemistry (IHC) analyses revealed that LiCl treatment inhibited GSK-3β activity in the 〖Olig2〗^+ OPCs/oligodendrocytes, confirming LiCl as a GSK-3β inhibitor in vivo. Moreover, LiCl treatment better preserved myelin and axons detected by myelin basic protein (MBP) immunostaining and neurofilment-200 (NF-200) immunostaining respectively in the injured spinal cords. All together, the data from our in vitro and in vivo experiments suggested that LiCl treatment after spinal cord injury is beneficial for functional recovery by preventing the loss of myelin and axons after SCI and this effect is mediated via GSK-3β inhibition This study provided evidence for the involvement of GSK-3β in the regulation of OPC differentiation and the subsequent remyelination in the injured adult spinal cord. We propose GSK-3β as an important therapeutic target for SCI repair, LiCl as a potential candidate for SCI clinical treatment and the possibility to manipulate endogenous NPCs after SCI to enhance oligodendrocyte differentiation, remyelination, and ultimately better functional recovery.. / published_or_final_version / Anatomy / Master / Master of Philosophy

Injuries to the central nervous system are irreversible and debilitating due to the limited regrowth of damaged or severed neurons. Two small molecules, xanthofulvin and vinaxanthone, isolated from P. vinaceum and P. glabrum promote spinal cord regeneration in animal models. It is speculated that these natural products inhibit semaphorin 3A, a chemorepellent that mitigates axonal growth-cone formation. In addition to promoting axonal growth, rats treated with vinaxanthone and xanthofulvin following complete spinal cord transection experienced greater remyelination, increased angiogenesis, attenuated apoptosis, and depressed scaring of the lesion site. The only prior synthesis of vinaxanthone speculated that the xanthone core is constructed via enzyme-catalyzed intermolecular Diels-Alder reaction. We have demonstrated, however, that warming a functionalized acetoacetyl chromone in water, furnishes vinaxanthone in good yield, providing an alternative biosynthetic pathway. With a robust syntheses of both natural products, we determined the protein target of the observed regeneration: succinate receptor 1, providing a new therapeutic target to promote neuronal regeneration. Among the various methods of incorporating oxygen into aryl rings, the direct conversion of a C-H bond into a C-OH bond is ideal. The metal-free hydroxylation of arenes developed in our laboratory, utilizing phthaloyl peroxide, marks the first disclosure of this transformation using mild conditions. Computational and experimental evidence obtained thus far has supported a mechanism involving a diradical intermediate. The reactivity of phthaloyl peroxide was increased by the incorporation of two chlorine atoms onto the ring. To minimize the accumulation of large quantities of peroxide, the optimization of the preparation of the peroxide in flow has been developed. This protocol immediately consumes the peroxide as it is generated. Finally, a new dearomatization reaction has been optimized. This reaction forms two carbon-oxygen bonds and dearomatizes the ring system.