Bone Material Quality, Structure, and Functional Relationships Contribute to Bone Strength and Toughness

Fragility fractures affect 8.9 million people annually, which is characterized by low bone mineral density (BMD) and deteriorating bone microarchitecture. Clinical diagnosis of people with fracture prone bones includes assessment of BMD via DEXA. However, BMD accounts for only 60% of the variation in bone fracture risk, since bone fracture risk comprises of bone strength and toughness. In addition to BMD, strength and toughness are influenced by bone material quality and bone microarchitecture as well. Additionally, these contributors to bone fracture risk are genetically controlled and are functionally related to each other. Thus, it was hypothesized that bone material quality, structure, and their functional relationships with strength and toughness, contribute to fracture risk. In aim 1, femurs from mice subjected to skeletal unloading in microgravity had reduced fracture resistance. Microgravity disrupted the ability of osteocytes to remodel their surrounding bone matrix, resulting deteriorated bone material heterogeneity, softened mineralized collagen fibrils, and weakened extrinsic toughening mechanisms. BMD, bone strength and toughness are all correlated, and these correlations called functional relationships are governed by genetics. However, functional relationships between bone microarchitecture and material quality have not been studied for genetically diverse group of mice. Additionally, how unloading affects the functional relationships is yet to be understood. Thus, in aim 2, it was hypothesized that functional relationships exist between bone material quality, bone structure and bone fracture risk across eight strains of mice with widely varying bone traits determined by genetics (i.e., in the Diversity Outbred, DO, founder strains), and these functional relationships become weaker with unloading. In aim 2, we found that functional relationships between bone microarchitecture, material quality and bone fracture risk exist for femurs of DO founder strains. Ratio of total area and bone length called slenderness ratio, bone material quality traits such as mineral:matrix, crystallinity and their heterogeneities contributed to the functional relationships and estimation of bone fracture risk. Following unloading, the functional relationships associated with strength and toughness become separate. Additionally, functional relationships after unloading were heavily guided by bone microarchitectural changes. Fracture resistance of bone is determined by its bone strength, i.e., stiffness, yield strength and bone toughness, i.e., resisting crack initiation and propagation. Intrinsic mechanisms represent the inherent resistance of bone material to elastic and plastic deformation via generating plasticity. Ribosylation of bones increases the advanced glycation end-products (AGEs) which increases non-enzymatic crosslinks and impair bone strength and toughness. Despite numerous studies, few studies have evaluated bone material quality assisted changes to tissue toughening mechanisms and risk of bone fracture with increased AGEs. Thus, in aim 3, it was hypothesized that bone material quality measures including tissue modulus (E), hardness (H), brittleness (H3/E2), tissue scratch toughness, material composition and material heterogeneity can explain changes to tissue toughening mechanisms that ultimately influence risk of bone fracture. In aim 3, we found that bones with ribosylation had reduced scratch toughness, tissue modulus, hardness and brittleness, along with poor macro-scale strength and toughness. These results provide insight into how bone material quality changes associated with tissue toughening mechanisms contributes to increased fracture risk with increased AGEs. Collectively, work presented in this dissertation will enable better understanding of role of bone material quality in influencing bone fracture risk.</p

Transcriptome-wide association study and eQTL colocalization identify potentially causal genes responsible for bone mineral density GWAS associations

Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni <= 0.05) using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.First author draf

Conventional and microwave-assisted synthesis of new 1H-benzimidazole-thiazolidinedione derivatives: A potential anticancer scaffold

A series of new benzimidazole bearing thiazolidinedione derivatives has been designed, synthesized by using conventional as well as microwave-assisted methods. Microwave-assisted synthesis caused a significant reduction in the reaction times and improvement in the yields of all the derivatives. All the new synthesized compounds were evaluated for their in vitro cytotoxic potential against selected human cancer cell lines of breast (MDAMB-231), prostate (PC-3), cervical (HeLa), lung (A549) and bone (HT1080) along with a normal kidney cells (HeK-293T). The compounds 17n, 17p and 17q were found to be potent cytotoxic with IC 50 values in the range of 0.096-0.63 μM on PC-3, HeLa, A549 and HT1080 cancer cells. Most of the compounds have found to be safe on normal HeK-293T kidney cells in comparison to cancer cells. The treatment of cells with 17p and 17q showed the typical apoptotic morphological features like fragmentation and shrinkage of nuclei. Further, test compounds resulted in inhibition of cell migration through disruption of F-actin protein assembly. Hoechst, DCFH-DA staining, mitochondrial membrane and annexin binding assays revealed that the cancer cell proliferation was inhibited through induction of apoptosis in A549 cells

Effect of Sulfamic Acid on 1,3-Dipolar Cycloaddition Reaction: Mechanistic Studies and Synthesis of 4-Aryl-NH-1,2,3-triazoles from Nitroolefins

A facile and new metal-free 1,3-dipolar cycloaddition reaction for the synthesis of 4-aryl-NH-1,2,3-triazoles from nitroolefins and NaN3 employing NH2SO3H has been developed. Sulfamic acid proved to be an efficient additive in this transformation by inhibiting the formation of triaryl benzene. Mechanistic aspects and key intermediates associated with this transformation have also been characterized by online monitoring of the reaction using electrospray ionization tandem mass spectrometry method (ESI-MS/MS). The protocol emphasizes broad substrate scope for many functionalities, simple reaction conditions such as stability to open air, less reaction time, easy work-up, eco-friendly and with good to excellent yields

New (E)-1-alkyl-1H-benzo[d]imidazol-2-yl)methylene)indolin-2-ones: Synthesis, in vitro cytotoxicity evaluation and apoptosis inducing studies

A new series of (E)-benzo[d]imidazol-2-yl)methylene)indolin-2-one derivatives has been synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines of prostate (PC-3 and DU-145) and breast (BT-549, MDA-MB-231, MCF-7, 4T1), non-small lung (A549) and gastric (HGC) cancer cells along with normal breast epithelial cells (MCF10A). Among the tested compounds, 81 showed significant cytotoxic activity against MDA-MB-231 and 4T1 cancer cells with IC50 values of 3.26 +/- 0.24 mu M and 5.96 +/- 0.67 mu M respectively. The compounds 8f, 8i, 8l and 8o were also screened on normal human breast epithelial cells (MCF10A) and found to be safer with lesser cytotoxicity. The treatment of MDA-MB-231 cells with 81 led to inhibition of cell migration ability through disruption of F-actin protein assembly. The flow-cytometry analysis reveals that the cells arrested in G0/G1 phase of the cell cycle. Further, the compound 81 induced apoptosis of MDA-MB-231 cells was characterized by different staining techniques such as Acridine Orange/Ethidium Bromide (AO/EB), DAPI, annexin V-FITC/PI, Rhodamine-123 and MitoSOX red assay. Western blot studies demonstrated that the compound 81 treatment led to activation of caspase-3, increased expression of cleaved PARP, increased expression of pro-apoptotic Bax and decreased expression of anti-apoptotic Bcl-2 in MDA-MB-231 cancer cells