Collagen and glycosaminoglycans, such as hyaluronan and chondroitin sulfate, are the major components of bone extracellular matrix, and extracellular matrix composites are being evaluated for a wide range of clinical applications. The molecular and cellular effects of native and sulfatemodified glycosaminoglycans on osteocytes were investigated as critical regulators of bone remodeling. The effects of glycosaminoglycans on viability, necrosis, apoptosis, and regulation of gene expression were tested in two osteocyte-like cell lines, the murine MLO-Y4 and the rat UMR 106-01 cells. Glycosaminoglycans were non-toxic and incorporated by osteocytic cells. In MLO-Y4 cells, sulfation of glycosaminoglycans led to a significant inhibition of osteocyte apoptosis, 42% inhibition for highly sulfated chondroitin sulfate and 58% for highly sulfated hyaluronan, respectively. Cell proliferation was not affected. While treatment with highly sulfated chondroitin sulfate increased cell viability by 20% compared to the native chondroitin sulfate. In UMR 106- 01 cells, treatment with highly sulfated hyaluronan reduced the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio by 58% compared to the non-sulfated form, whereas highly sulfated chondroitin sulfate led to 60% reduction in the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio in comparison to the native chondroitin sulfate. The expression of SOST, the gene encoding sclerostin, was reduced by 50% and 45% by highly sulfated hyaluronan and chondroitin sulfate, respectively, compared to their native forms. The expression of BMP- 2, a marker of osteoblast differentiation, was doubled after treatment with the highly sulfated hyaluronan in comparison to its native form. In conclusion, highly sulfated glycosaminoglycans inhibit osteocyte apoptosis in vitro and promote an osteoblast-supporting gene expression profile.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (STAT3) is known to be related to bone metabolism. Mutation of STAT3 causes a rare disorder in which serum levels of IgE are elevated. This causes various skeletal problems similar to osteoporosis. To examine the effect of STAT3 in the osteoclast, we obtained two osteoclast specific STAT3 knockout mouse models: one using the CTSK promoter to drive Cre recombinase and another using a TRAP promoter. Examination of these mice at 8 weeks of age revealed a decreased trabecular bone volume in CTSK specific STAT3 knockout mice along with a slight decrease in osteoclast number in both CTSK and TRAP specific STAT3 knockout females. We also noticed changes in bone mineral density and bone mechanical strength in females. These data suggest that STAT3 plays a part in the function of the osteoclast.

Indiana University-Purdue University Indianapolis (IUPUI) / An excess of bone resorption over bone formation leads to osteoporosis, resulting in a reduction of bone mass and an increase in the risk of bone fracture. Anabolic and anti-resorptive drugs are currently available for treatment, however, none of these drugs are able to both promote osteoblastogenesis and reduce osteoclastogenesis. This thesis focused on the role of eukaryotic translation initiation factor 2 alpha (eIF2alpha), which regulates efficiency of translational initiation. The elevation of phosphorylated eIF2alpha was reported to stimulate osteoblastogenesis, but its effects on osteoclastogenesis have not been well understood. Using synthetic chemical agents such as salubrinal and guanabenz that are known to inhibit the de-phosphorylation of eIF2alpha, the role of phosphorylation of eIF2alpha in osteoclastogenesis was investigated in this thesis. The questions addressed herein were: Does the elevation of phosphorylated eIF2alpha (p-eIF2alpha) by salubrinal and guanabenz alter osteoclastogenesis? If so, what regulatory mechanism mediates the process? It was hypothesized that p-eIF2alpha could attenuate the development of osteoclast by regulating the transcription factor(s) amd microRNA(s) involved in osteoclastogenesis. To test this hypothesis, we conducted in vitro and in silico analysis of the responses of RAW 264.7 pre-osteoclast cells to salubrinal and guanabenz. First, the in vitro results revealed that the elevated level of phosphorylated eIF2alpha inhibited the proliferation, differentiation, and maturation of RAW264.7 cells and downregulated the expression of NFATc1, a master transcription factor of osteoclastogenesis. Silencing eIF2alpha by RNA interference suppressed the downregulation of NFATc1, suggesting the involvement of eIF2alpha in regulation of NFATc1. Second, the in silico results using genome-wide expression data and custom-made Matlab programs predicted a set of stimulatory and inhibitory regulator genes as well as microRNAs, which were potentially involved in the regulation of NFATc1. RNA interference experiments indicated that the genes such as Zfyve21 and Ddit4 were primary candidates as an inhibitor of NFATc1. In summary, the results showed that the elevation of p-eIF2alpha by salubrinal and guanabenz leads to attenuation of osteoclastogenesis through the downregulation of NFATc1. The regulatory mechanism is mediated by eIF2alpha signaling, but other signaling pathways are likely to be involved. Together with the previous data showing the stimulatory role of p-eIF2alpha in osteoblastogenesis, the results herein suggest that eIF2alpha-mediated signaling could provide a novel therapeutic target for treatment of osteoporosis by promoting bone formation and reducing bone resorption.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Dicalcium phosphate dihydrate (DCPD) cements have been used for bone repair due to its excellent biocompatibility and resorbability. However, DCPD cements are typically weak and brittle. To overcome these limitations, the sodium citrate used as a setting regulator and the coating of poly-L-lactide acid (PLLA) technique have been proposed in this study. The first purpose of this thesis is to develop composite PLLA/DCPD scaffolds with enhanced toughness by PLLA coating. The second purpose is to examine the biocompatibility of the scaffolds. The final purpose is to investigate the degradation behaviors of DCPD and PLLA/DCPD scaffolds. In this experiment, DCPD cements were synthesized from monocalcium phosphate monohydrate (MCPM) and 𝛽-tricalcium phosphate (𝛽 –TCP) by using deionized water and sodium citrate as liquid components. The samples were prepared with powder to liquid ratio (P/L) at 1.00, 1.25 and 1.50. To fabricate the PLLA/DCPD composite samples, DCPD samples were coated with 5 % PLLA. The samples were characterized mechanical properties, such as porosity, diametral tensile strength, and fracture energy. The mechanical properties of DCPD scaffolds with and without PLLA coating after the in vitro static degradation (day 1, week1, 4, and 6) and in vitro dynamic degradation (day 1, week 1, 2, 4, 6, and 8) were investigated by measuring their weight loss, fracture energy, and pH of phosphate buffer solution. In addition, the dog bone marrow stromal stem cells (dBMSCs) adhesion on DCPD and PLLA/DCPD composite samples were examined by scanning electron microscopy. The cell proliferation and differentiation in the medium conditioned with DCPD and PLLA/DCPD composite samples were studied by XTT (2,3-Bis(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt), and alkaline phosphatase (ALP) assay, respectively. The addition of sodium citrate and PLLA coating played a crucial role in improving the mechanical properties of the samples by increasing the diametral tensile strength from 0.50 ± 0.15 MPa to 2.70 ± 0.54 MPa and increasing the fracture energy from 0.76 ± 0.18 N-mm to 12.67 ± 4.97 N-mm. The DCPD and PLLA/DCPD composite samples were compatible with dBMSCs and the cells were able to proliferate and differentiate in the conditioned medium. The degradation rate of DCPD and PLLA/DCPD samples were not significant different (p > 0.05). However, the DCPD and PLLA/DCPD composite samples those used sodium citrate as a liquid component was found to degrade faster than the groups that use deionized water as liquid component