Doctor of Philosophy, University of Akron, 2019, Biomedical Engineering

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is an elusive disease that presents as exposed necrotic bone following tooth extraction. It occurs in patients undergoing bisphosphonate therapy for metastasizing cancers and osteoporosis. Experts believe the condition is caused by a defect in bone remodeling, the process by which osteoclasts resorb bone and osteoblasts form new bone, within the oral cavity. Its complexity requires a multicellular model to address the net effects of two key risk factors: tooth extraction (overload) and inflammation associated with bacterial infection. In this work, a system comprised of a deformable polymeric chip and mechanical loading device is used to expose bisphosphonate-treated osteocytes, the mechanosensing bone cells, to overload. Osteocyte viability is evaluated as a function of load, and soluble activity is assessed. Effects of these factors on bone resorption by osteoclasts and bone formation by osteoblasts are quantified. Osteoclast activity is also quantified in the presence of inflammatory agents, lipopolysaccharide and interferon gamma. Results support a role for osteocyte mechanotransduction in suppressing osteoblast bone formation within a BRONJ environment. They also suggest inflammation may inhibit resorption of necrotic bone by osteoclasts. These findings provide insights into BRONJ that may contribute to its elucidation. This dissertation also lays the foundation for a biomimetic lab-on-a-chip platform for the study of bone turnover and remodeling-related disease. Fabrication techniques are developed, and osteocyte, osteoclast and osteoblast characterizations are performed on relevant substrates within microfluidic devices. Culture conditions, including seeding densities, feeding requirements and time points for analyses are determined. This work will enable the development of a controlled multicellular lab-on-a-chip capable of quantifying the aggregate response of bone cells to disease cofactors.

Committee: Marnie Saunders PhD (Advisor); Hossein Tavana PhD (Committee Member); Ge Zhang PhD (Committee Member); Jiang Zhe PhD (Committee Member); Sailaja Paruchuri PhD (Committee Member) Subjects: Biomedical Engineering

MS, Kent State University, 2016, College of Arts and Sciences / School of Biomedical Sciences

Trafficking Protein Particle Complex 9 (TRAPPC9) is a major subunit of TRAPP Complex. TRAPPC9 has been reported to bind IKappaB Kinase (IKK2) NF-KappaB-inducing kinase (NIK) where it plays a role in the canonical and non-canonical of NFkB signaling in Osteoclast (OC) differentiation and function. Respectively, the role of TRAPPC9 in protein trafficking in OC has not been studied. In this study, we first examined the co-localization of TRAPPC9 with cathapsin-K (Cathp.K), known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking and function of OCs. Second, to identify TRAPPC9 protein partners important for OC-mediated bone resorption, we conducted immunoprecipitation of TRAPPC9 isolated from terminally differentiated OCs followed by mass spectrometry analysis. Surprisingly, our data showed that TRAPPC9 binds various protein partners. One of those proteins is L-Plastin (LPL). LPL is localized at the podosomes and plays a crucial role in actin aggregation thereby actin ring formation and OC function. Recent studies reported that LPL null OCs demonstrated normal OC differentiation phenotype and peripheral podosomes aggregation. However, significant disruption in actin ring formation and the sealing zone region were observed. Although the role of LPL in OC-mediated bone resorption has not reported in details. Here, we investigated the potential regulatory role of TRAPPC9 and LPL-mediated OC differentiation and function. Thereby, we assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is co-localized with LPL within the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression using viral system on LPL recruitment to the actin ring. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared to controlled cultures. This recruitment is associated with increasing OC-mediated bone resorption. In conclusion, our hypothesis is that TRAPPC9 plays a regulator (open full item for complete abstract)

Committee: Fayez Safadi (Advisor) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Cathepsin K (CatK), a cysteine protease, has been implicated in the process of bone resorption and inflammation. Selective inhibitors of CatK are promising therapeutic agents for the treatment of diseases associated with excessive bone loss and osseous inflammation, such as osteoarthritis, rheumatoid arthritis, periodontitis, osteoporosis, and multiple myeloma. Multiple reports have emerged over the last several years demonstrating the effect of different CatK inhibitors on osteo-inflammatory conditions. Therefore, the study of CatK inhibition as a target to prevent bone loss and inflammation, and influence bone marrow osseous progenitor cells, in a large animal model, is the subject of this dissertation. The horse was selected as the large animal model because this species suffers from ailments of adaptive bone remodeling in their sport performance and CatK inhibitors may serve as therapeutics in this species as well as serve as a large animal model for human applications. In the first phase of this work, we determined an optimal dose and dose interval for a CatK inhibitor (CatKI), VEL-0230, in healthy adult horses. Plasma pharmacokinetic (PK) and bone resorption biomarker [carboxy-terminal cross-linking telopeptide of type I collagen (CTX-1)] analyses were performed following single and multiple oral dose protocols of a CatKI (VEL-0230) in horses. Weekly administration of VEL-0230, at a dose of 4 mg/ kg body weight, provided effective inhibition of bone resorption in young exercising horses that returned to baseline within 7 days after drug withdrawal even after multiple doses. In the second phase of this work, we evaluated bone structure and turnover in healthy young exercising horses receiving repeated oral dosing of a CatKI in a randomized, controlled, double-blinded, prospective, sufficiently powered clinical trial. With the objectives of: 1. To determine whether repeated dosing of a CatKI produced a desired inhibition of the bone resorption biomarker (open full item for complete abstract)

Committee: Alicia Bertone PhD (Advisor); Maxey Wellman PhD (Committee Member); Prosper Boyaka PhD (Committee Member); Teresa Burns PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology

PHD, Kent State University, 2015, College of Arts and Sciences / School of Biomedical Sciences

Osteoporosis impacts 55% of the population aged 50 and older, with post-menopausal, thin women of Caucasian descent at the highest risk for developing this disease. As the median age of the population rises, so does the incident of fracture caused by osteoporosis. Currently, only one bone anabolic factor is available on the market, PTH1-34, with a therapeutic window of only 2 years; new novel bone anabolic factors are needed. One protein with the potential for osteoporosis therapy is osteoactivin (OA). Previous studies have demonstrated the role of OA in osteoblast and osteoclast differentiation and function. In this study, we utilized global OA knockout mice to explore the physiological role of OA in bone homeostasis. We also characterized the role of osteoactivin in osteoblasts and osteoclasts by studying OA-/- cells ex vivo. In addition, we tested how the skeleton responded to stress induced by estrogen-deficient osteoporosis model in OA-mutant (D2J), OA knockout, and their respective wildtype controls. Additionally, we investigated the functional role of osteoactivin-derived peptide OA-D. We hypothesized that osteoactivin was critical for postnatal bone growth and development, and that OA-D would be capable of stimulating bone formation and rescuing osteoporotic phenotypes. We demonstrated that the OA-null animals had increased bone mass. Ex vivo analysis of osteoblast differentiation and function revealed defects in mineralization and differentiation. Next, we observed that osteoclast size due to increased fusion of large osteoclasts was significantly increased in the OA-/- animal. However, osteoclast resorption was significantly decreased. Next, OA-D was tested in C57/Blk6 mice for its ability to stimulate bone mass. We showed that OA-D injection in mice resulted in a trending increase in bone mass. Alternative administration of OA-D showed similar results in bone mass. Ovariectomy of OA mutant and OA-/- animals failed to induce significant bone loss (open full item for complete abstract)

Committee: Fayez Safadi PhD (Committee Chair); Werner Geldenhuys PhD (Committee Member); Chris Malcuit PhD (Committee Member); Moses Oweyumi PhD (Committee Member); Walt Horton PhD (Committee Member); Srinivasan Vijayaraghavan PhD (Committee Member) Subjects: Biology; Cellular Biology; Pharmacology

PhD, University of Cincinnati, 2001, Medicine : Molecular, Cellular and Biochemical Pharmacology

Osteoporosis leads to fracture and a simultaneous increase in mortality from such injuries. Individuals over the age of 50 are especially prone to osteoporosis, in part due to their age and the resulting deficit in sex steroid hormones. Androgen deficiency in males leads to an increase in osteoclastic bone resorption and a progressive decrease in bone mineral density. Demineralization in key structural regions of bone reduces skeletal integrity and increases the risk of fracture, analogous to that observed in females following menopause. Therefore, androgens may play a similar role to estrogens in the direct regulation of the bone resorbing cell, the osteoclast. Steroid control over osteoclast differentiation and proliferation may help prevent the onset of age-related osteoporosis. We examined the ability of testosterone (T) and 5α-dihydrotestosterone (5α-DHT) to suppress osteoclast formation induced by receptor activator NF-κB (RANK) ligand and macrophage colony stimulating factor (M-CSF) in vitro. Androgens suppressed the osteoclast formation from bone marrow monocytes (BMMs) of both normal and orchidectomized mice. Interestingly, 17β-estradiol was as effective as both T and 5α-DHT in suppressing osteoclast formation in BMMs from both normal and orchidectomized mice. A similar sensitivity to both sex hormones was observed following ovariectomy. As with BMMs, T and 5α-DHT also suppressed osteoclast formation in RAW264.7 cells indicating that the suppressive effects of androgens on osteoclastogenesis are direct and do not require stromal cells. In RAW264.7 cells, androgens inhibit RANKL-induced osteoclast formation through selective regulation of c-Jun. Increases in precursor number following orchidectomy led us to also examine the ability of the sex steroids to inhibit proliferation of these cells. Both androgens and estrogens appear to inhibit M-CSF- mediated proliferation of both BMM and RAW264.7 cells. The proliferative effect was due in part to upregulation of (open full item for complete abstract)

Committee: J. Pike (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2012, Integrated Biomedical Science Graduate Program

Osteoporosis is a systemic metabolic bone disorder characterized by low mineral density and micro architectural deterioration of skeleton. Osteoporosis or “porous bone” disease can progress asymptomatically until bones fracture. It is a major cause of morbidity and health care burden worldwide. Lack of physical activity, hormonal disturbances, age, gender, heredity and nutritional deficiency are some the factors implicated in causing the disease. Physiologically, bone is a dynamic organ that constantly undergoes remodeling. Bone consists of dense organic and inorganic components. At cellular level, balance/coupling between the activities of bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts) maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Several therapies such as hormone replacement therapy in women, administration of anti-resorptives (bisphosphonates), implantation of osteoinductive device combined with change in lifestyle offers only temporary augmentation of bone mineral density with side effects. Consequently, the potential of stem cell based therapies is currently being considered. Adult stem cells expressing CD133/CD34 surface marker are multipotent cells that can be isolated from human umbilical cord blood and differentiated into many cell lineages by changing the molecular cues in their microenvironment. However, their limited numbers from a single unit restricts their experimental use. Our lab developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated for experimental use and potential therapeutic applications. Herein, we show that nanofiber-expanded CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, BMD and bone micro-architecture in osteoporotic aged nono (open full item for complete abstract)

Committee: Hiranmoy Das Dr./Ph.D. (Advisor); Periannan Kuppusamy Dr./Ph.D. (Committee Member); Sudha Agarwal Dr./Ph.D. (Committee Member); Nancy Lill Dr./Ph.D. (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2008, Molecular, Cellular, and Developmental Biology

Osteoclasts perform bone resorption vital to the maintaining of healthy bone and blood calcium levels. Osteoclasts function by forming unique actin attachment structures for migration (podosomes) and bone resorption (actin rings). For these studies, the role of two actin binding protein classes, tropomyosins and myosins, are studied to reveal their function in osteoclast differentiation and podosome/actin ring dynamics. Our research initially demonstrated the presence of nine tropomyosin isoforms in distinct locations in osteoclasts. Tm4 co-localized with the interior faces of actin rings and podosomes. RNAi-mediated suppression led to reductions in actin ring thickness, bone resorption, and motility. Overexpression of exogenous Tm4 resulted in abnormal, thicker podosomes that were unusually distributed along with reduced cell motility and abolished bone resorption. We hypothesize that the main role of Tm4 is regulation of the adhesion structures of osteoclasts by stabilizing filamentous actin in podosomes and actin rings and thus affecting migration and bone resorption. Myosins are a class of molecular motors that provide the ATP-dependent force to generate movement such as vesicle transport, cell migration, and cell division. Our previous studies showed MyoIIA to be distributed within podosomes and the actin ring of polarized osteoclasts. During differentiation, MyoIIA protein levels temporarily diminished corresponding to fusion initiation. Cathepsin B regulated the temporary protein decrease. RNAi of MyoIIA was used during the latter stages of osteoclastogenesis. This suppression generated very large, less motile osteoclasts that were a result of increased fusion (elevated numbers of nuclei per cell) and cell spreading (perimeter per nuclear number). While the large cells, which formed large actin rings, were capable of resorbing bone, their diminished motility resulted in minimal changes in resorption. These data suggest MyoIIA plays an inhibitory role during o (open full item for complete abstract)

Committee: Beth Lee PhD (Advisor); Anthony Brown PhD (Committee Member); Harold Fisk PhD (Committee Member); Arthur Strauch PhD (Other) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Pharmacology

The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. In turn, these gene products mediate the actions of 1,25(OH)2D3 in mineral-regulating target cells. 1,25(OH)2D3 impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct regulatory roles of 1,25(OH)2D3 in bone, relatively little is known about the mechanisms or 1,25(OH)2D3-target genes in skeletal cells. Here, we identified meningioma 1 (MN1) and semaphorin 3B (SEMA3B) as two novel 1,25(OH)2D3-stimulated genes in osteoblastic cells, and uncovered their new functions in skeletal physiology. We demonstrated that MN1 is a coactivator for VDR-mediated transcription, and calvarial osteoblasts derived from MN1 knockout mice displayed altered morphology, decreased growth rate, impaired motility, attenuated 1,25(OH)2D3/VDR-mediated transcription, reduced alkaline phosphatase activity, decreased mineralized nodule formation, but enhanced adipogenesis. In addition, MN1 knockout osteoblasts are defective in supporting 1,25(OH)2D3-stimulated osteoclastogenesis, presumably due to marked reduction in the RANKL:OPG ratio. These data indicate an important role for MN1 in maintaining appropriate osteoblast proliferation, maturation and function. This may partially account for the intramembranous ossification defects of cranial bones in MN1 knockout mice. Our data reveal that osteoblast-derived SEMA3B alters the global skeletal homeostasis in intact animals and the bone cell activities in cultures. Transgenic mice with osteoblast-targeted over-expression of SEMA3B develop osteopenia, with decreased body weight, reduced bone mineral density, and aberrant trabecular structure compared to the nontransgenic littermates. Histomorphometry studies indicated that this was (open full item for complete abstract)

Committee: Ruth Keri (Committee Chair); Paul MacDonald (Advisor); Diane Dowd (Committee Member); Yu-Chung Yang (Committee Member); David Danielpour (Committee Member) Subjects: Pharmacology