Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.

Nanoindentation techniques have not been standardized for use on bone tissues, making comparison of bone material properties obtained via nanoindentation across studies difficult and unreliable. This study determined a set of optimal parameter values for thermal drift correction time, dwell time, and loading rate that can be used to obtain accurate and repeatable material properties from human femoral trabecular bone through experimentation and statistical analysis. All testing was conducted using a single nanoindenter on a single trabeculae, with the assumption that material properties within the individual trabeculae were internally consistent. Parameters not of interest during this study, such as ambient temperature, maximum load, and maximum indentation depth were held constant throughout all experiments. Elastic modulus and hardness data were calculated using the Oliver-Pharr technique. The optimal values for these parameters are as follows: 150 seconds for thermal drift correction time, 30 to 60 seconds for dwell time, and 0.4 to 0.8 mN/s for loading rate.

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Bone dysplasia’s affect every 1 in 5,000 babies; most of these dysplasia’s are incurable, and some are even lethal (Stembalska et al., 2021). Hundreds of skeletal dysplasia’s are heritable, yet the genes involved are not well defined (Krakow, 2015). Most of the skeleton forms through a process called endochondral ossification (EO). There are three parts of EO: chondrogenesis, maturation, and ossification. During chondrogenesis, mesenchymal progenitor cells condense and then differentiate into chondrocytes. After differentiation, chondrocytes will elongate, then proliferate and mature to set up for primary ossification. We know that this process happens through many activated genes, but the sequential steps through which this is achieved has yet to be elucidated. In order to understand the cause of skeletal dysplasia’s and find new treatments, the molecular mechanisms controlling EO requires further investigation. This study focuses on one gene, CXXC1 Finger Protein, Cfp1, and its role in chondrocyte maturation during skeletal mouse development. Cfp1 was specifically deleted in chondrocytes, and the resultant effects on cartilage and bone were analyzed. A mild phenotype was observed in the knockout mouse model. It was found that loss of Cfp1 in chondrocytes leads to delayed ossification in the vertebrae, tibias, metatarsals, and metacarpals. Therefore, Cfp1 is necessary for normal chondrocyte maturation.

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Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. Recently, the combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance has become a growing concern. Current evidence points towards a chemoprotective role from the bone marrow (BM) microenvironment, specifically by BM-derived mesenchymal stromal cells (BMSCs) and adipocytes. AML cells can manipulate BMSCs and adipocytes to create a niche that supports its own growth and evades chemotherapy. However, the role of the microenvironment in Ven/Aza chemoresistance has yet to be studied. Our objective was to study the ability of the microenvironment cells to induce AML chemoresistance to Ven/Aza. We employed a 2-dimensional direct contact co-culture system between MOLM-13 AML cells and BMSCs or adipocytes in both the absence and presence of Ven/Aza to determine the effects on the AML cells. In the absence of Ven/Aza, adipocyte co-cultured AML cells showed a 47% reduction in proliferation, 10% reduction in viability, yet a 1.7-fold increase in Maximal respiration when compared to the monocultured cells. In the presence of Ven/Aza, adipocyte co-cultured AML cells showed a significant increase in both proliferation and viability. Preliminary work investigating the mechanism of action of this support points toward an anti-apoptotic mechanism mediated by the upregulation of MCL-1 upon co-culture with adipocytes. Combination of Venetoclax and Tapotoclax, an MCL-1 inhibitor, abrogated the chemoprotection provided by BMSCs and adipocytes. Overall, our data suggests a dual role of adipocytes, where their inhibition or support of AML is context dependent. Therapeutic targeting of mechanisms for adipocyte chemoprotection such as MCL-1 upregulation may re-sensitize AML cells to Ven/Aza, thereby improving patient outcomes. / Thesis / Master of Science (MSc) / Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. A novel combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance remains an important concern. Previous studies have implicated fat cells, or adipocytes, in AML chemoresistance, however, their role in Ven/Aza treatment has yet to be studied. Here, we show that adipocytes reduce growth of AML cells, yet enhance their metabolism. In the presence of Ven/Aza, adipocytes induce chemoresistance. We show preliminary data that this chemoprotection may be mediated by the upregulation of mitochondrial MCL-1 protein as inhibition of this protein neutralized the protection. By understanding the relationship between adipocytes and AML chemoresistance, we can target this and re-sensitize AML to Ven/Aza, thereby improving older patient outcomes.