Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

It is well established that bone is capable of adapting to changes in its environment; however, little is known regarding how environmental stimuli, specifically loading, are associated with the internal 3D microarchitecture of cortical bone. The aim of this thesis was to develop a small animal model that can be used to experimentally test hypotheses regarding bone adaptation. High resolution micro-CT was validated and employed as a novel method for the visualization and quantification of rat cortical bone microarchitecture in 3D. The use of this imaging method allowed for the measurement of primary vascular canal orientation in 3D, which had never been achieved before. Using this measure along with an immobilization model for unloading allowed me to test how loading is associated with the orientation of these vascular canals. Normally ambulating rat bones (from 10 female rats) had a canal structure that was 9.9° more longitudinal than their immobilized counterparts. This finding that loading has an effect on primary canal orientation brought to light the need to induce remodeling and therefore, secondary vascular canals, in the rat to increase its novelty as a model for looking at bone adaptation. Remodeling was induced by increasing the calcium demands of female rats, either through a calcium restricted diet (n=2) or pregnancy and lactation coupled with a calcium restricted diet (n=2). Mean cortical thickness for the calcium restricted rats and the pregnant and lactating rats that were on a calcium restricted diet were 622 µm and 419 µm, respectively. The mean BMU count for calcium restricted rats seemed to be higher than that of the pregnant and lactating rats; however, the calcium restricted rats seemed to have a lower BMU density. Once this full-scale study is executed the rat will provide a more representative model for studying human bone adaptation.

Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

Across vertebrates, the coordinated evolution and synchronous development of teeth and the mandible must require specific timing and positioning of gene expression. While debate persists about whether teeth have evolved before or after mandible, currently, the consensus is that these systems evolved at separate times and thus have discreet origins. This raises an important question of whether tooth and mandibular tissues have over the course of their evolution become developmentally co-dependent or, as separate evolutionary origins would imply, remain developmentally autonomous of each other. The molecular signaling that patterns the genesis of upper versus lower jaw skeletons, as well as specifies tooth type (i.e., molar vs. incisor) is relatively well understood. To date, the distinct genetic processes that drive tooth development distinct from jaw skeletal development has been little-studied, in no small part due to the technical complexity of this task. The main hypothesis of thesis is that a collection of genes acting within a gene regulatory network (GRN) drives odontogenesis with neither input from, nor influence on, jaw morphogenesis. The Transformation Related Protein (TRP63) is a master transcription factor that is vital to odontogenesis because TRP63 maintains the competence and proliferation of the epithelial layer of the tooth organ. Thus the “toothless” TRP63 homozygote mouse mutant (Brdm2 mutant) fails to develop teeth even though it develops a virtually unperturbed mandible. This combination of lower jaw morphogenesis in the absence of odontogenesis presents a rare model to study the genetic changes that occur when teeth but not jaws fail to form. A previous microarray gene expression analysis (Boughner laboratory, unpublished data) of mandibular prominences (MdPs) derived from embryonic day (E) 10-13 revealed that, compared to heterozygote (Trp63+/-) MdPs, in Brdm2 mutant MdPs, transcript levels of cerebellin 1 (Cbln1); keratin 2-8 (Krt2-8); phospholipid transfer protein (Pltp) and fermitin 1 (Fermt1) were altered in at least some of the four embryonic stages. Specifically Cbln1 and Krt2-8 were up-regulated while Pltp and Fermt1 were down-regulated. None of these four genes have previously been linked to odontogenesis yet all are potential candidates for a “tooth-specific” GRN. Using RT-qPCR analysis, I aimed to test the validity of the microarray work and confirmed its veracity by showing that, generally, Cbln1 and Krt2-8 mRNA were up-regulated, while Fermt1 (but not Trp63 or Pltp) mRNA was significantly down-regulated in the MdPs of Brdm2 mutant mice relative to Trp63+/- mice. Conversely, western blotting protein expression analysis showed little-to-no change among Brdm2 MdPs relative to either wild type (Trp63 +/+) or Trp63+/- embryos, making it difficult to tease out the precise relationship between CBLN1, FERMT1, KRT2-8, and PLTP and TRP63. These results show a lack of strong correlation between mRNA and protein expression. Because the mRNA analyses showed disturbances in the expression level in a few of these five genes within the MdPs during the earliest stages of tooth development, these genes remain candidates for an odonto-specific GRN. In complement to the genetic work, to characterize the tandem developmental morphology of tooth and jaw skeleton tissues, my work included developing a new tissue staining protocol. Using Protargol, a silver-based compound, to enhance in uncut mouse embryos contrast among tiny, soft oral tissues and visualize their organization in 3D and microscopic detail across several embryonic stages. This novel protocol offers a simple, easy-to-follow, and relatively inexpensive way to effectively stain whole embryos aged E10-15 for X-ray based micro-computed tomography (μ-CT) imaging using synchrotron and desktop scanning systems. Because the scan data are digital, this new method also allows more precise, accurate and rapid empirical studies of the sizes, shapes and positions of teeth as they form within the jaw to clarify how these tissues are integrated as they develop. The work presented in this thesis investigated tooth development exclusive of mandible development from complementary molecular and morphological points of view. Driven by the lack of understanding of the genetic mechanisms that orchestrate tooth with jaw skeletal development, this study has for the first time isolated a set of genes that are potential candidates for tooth formation only. These results set the stage for next steps in testing the developmental-genetics that enable teeth and jaws to “fit” together as they develop.

Cortical bone is a dynamic tissue which undergoes adaptive and pathological changes throughout life. An improved understanding of the spatio-temporal process of remodeling holds great promise for improving our understanding of bone development, maintenance and senescence. The use of micro-computed tomography (µCT) on living animals is relatively new and allows the three dimensional quantification of change in trabecular bone microarchitecture over time. The use of in vivo µCT is limited by the radiation dose created by the x-ray beam, with commercially available in vivo systems generally operating in the 10-20 um resolution range and delivering an absorbed dose between 0.5-1 Gy. Because dose scales to the power of four with resolution, in vivo imaging of the cortical canal network, which requires a higher resolution, has not been achieved. I hypothesized that using synchrotron propagation phase contrast µCT, cortical porosity could be imaged in vivo in rats at a dose on the same level as those used currently for trabecular bone analysis. Using the BMIT-BM beamline, I determined the optimal propagation distance and used ion chamber and lithium fluoride crystal thermoluminescent dosimetry to measure the absorbed dose of my in vivo protocol as well as several ex vivo protocols using synchrotron phase contrast µCT at 5 µm, 10 µm, and 11.8 µm and conventional desktop in vivo protocols using commercial µCT systems. Using synchrotron propagation phase contrast µCT, I scanned the forelimb of two adult Sprague-Dawley rats and measured an absorbed dose of 2.53 Gy. Using two commercial µCT system, I measured doses between 1.2-3.6 Gy for protocols at 18µm that are in common use. This thesis represents the first in vivo imaging of rat cortical porosity and demonstrates that an 11.8 µm resolution is enough to visualize cortical porosity in rats, with a dose within the scope of those used for imaging trabecular bone in vivo.

In the area of translational research, stent developers consult pathologists to obtain the best and most complete amount of data from implanted test devices in the most efficient manner. Through the use of micron-scale computed tomography along with post-fixation staining techniques in this study, full volumes of previously implanted stents have been analyzed in-situ in a non-destructive manner. The increased soft tissue contrast imparted by metal-containing stains allowed for a qualitative analysis of the vessel’s response to the implant with greater sensitivity and specificity while reducing beam-hardening artifact from stent struts. The developed staining techniques included iodine-potassium iodide, phosphomolybdic acid, and phosphotungstic acid, all of which bind to soft tissue and improve image quality through their ability to attenuate high energy X-rays. With these stains, the overall soft tissue contrast increased by up to 85 percent and contrast between medial and neointimal layers of the vessel increased by up to 22 percent. Beam hardening artifact was also reduced by up to 38 percent after staining. Acquiring data from the entirety of the stent and the surrounding tissue increased the quality of stent analysis in multiple ways. The three dimensional data enabled a comprehensive analysis of stent performance, lending information such as neointimal hyperplasia, percent stenosis, delineation of vessel wall layers, stent apposition, and stent fractures. By providing morphological data about stent deployment and host response, this method circumvents the need to make the more traditional histology slides for a morphometric analysis. These same data may also be applied to target regions of interest to ensure histology slides are cut from the optimal locations for a more in-depth analysis. The agents involved in such techniques are readily available in most pathology laboratories, are safe to work with, and allow for rapid processing of tissue. The ability to forego histology altogether or to highly focus what histology is performed on a vessel has the potential to hasten the development process of any coronary stent.

Resin composites have been in the dental field for over forty years. They are now thought to be the most commonly used restorative material due to their aesthetic and mechanical properties. Although resin composites have high success rates as restorations, they do not offer all properties of an ideal restorative material. The aims of this research were to characterise the effects of variation in resin composite formulation on handling, mechanical; and physical properties. In particular the influence of the size and distribution of the inorganic components was investigated through the study of experimental formulations. Packing stress and viscosity were assessed with pentrometer principle at two different temperatures (23 and 37 ºC). It was found that filler size was strongly correlated with both packing stress and viscosity. Additionally, temperature has a dominant effect on packing stress and viscosity. Micro computed tomography [μCT] was used to investigate percentage of voids [% voids] in 3D dimensions. It was found that smaller filler size incorporated less % voids. In contrast filler size and disruption had a little effect on fracture toughness of resin composites. 3D surface topography was used to investigate the surface roughness before and after tooth brush abrasion. It was found filler size had a significant influence in both gloss retention and surface roughness (smaller filler size exhibited higher surface gloss). Finally, the effect of different storage media (distilled water, Coca Cola and red wine) on colour stability and gloss were investigated. It was found that dietary habits effect discolouration of resin composite restorations with the acidic drinks caused more staining.

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Objectives: While bracket debonding has been explored through shear bond-strength tests with debonding linked to the type of material used, the technique of adhesive application, and contamination, the contribution of internal voids in orthodontic adhesives is unknown. Voids may result in fracture or bond-failure, either within the adhesive or at the tooth-adhesive-bracket interfaces. The aim of this thesis is to quantify the internal volumetric voids and bonding strength of three generations of bracket adhesives. Methods: Extracted third molars were bonded with three groups of orthodontic brackets including conventionally-pasted (CP), pre-coated (PC), or pre-coated flash-free (FF) (n=5 per group). The three-dimensional internal structure of the adhesive was evaluated with Micro Computed Tomography (micro-CT) using the Skyscan micro-CT (maximum resolution of 5 microns). Data from the micro-CT were analyzed with SkyScan software to perform 3D reconstructions, image processing, and qualitative and quantitative analysis of the adhesive’s structure. The amount of void was determined by measuring the percentage of voids at the bonded interfaces (Vint) and within the adhesive (Vbulk). The total amount of void was also calculated (Vtot= Vint+ Vbulk). Differences in void between the groups were assessed using one-way ANOVA with post-hoc Tukey tests (α=0.05). The bonding strength of the three adhesives systems was evaluated via shear bond strength tests. Results: Our void quantification results showed that FF brackets had a statistically higher (p0.05) for any the analyzed locations (Vint, Vbulk). Our results indicate the majority of voids were found at bonded interfaces (Vint) compared to within the bulk (Vbulk) for all three groups, with statistically significant (p<0.05) differences for CP and FF. Our bonding strength evaluation revealed the pre-coated group (PC) to have on average the highest bond strength compared to conventionally-pasted (CP) and pre-coated flash-free (FF) groups. Conclusions: The overall amount of void in the pre-coated flash-free adhesive brackets is significantly higher compared to conventionally-pasted and pre-coated groups. The majority of the void was identified to be located at the bonded interfaces (tooth/adhesive and bracket/adhesive) rather than within the bulk of the adhesive for all groups. Our bonding strength results indicate the pre-coated brackets to have the highest bond strength compared to pre-coated flash-free and conventionally-pasted. Our preliminary set of results indicate an inverse relationship between bonding strength and amount of void of brackets adhesives, with the pre-coated exhibiting the highest bond strength and least void. However, due to a limited sample size additional data are needed to validate these conclusions and find solid relationships between adhesive voids and bonding strength. / Oral Biology