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

Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine

Prostate cancer (PCa) is the most common malignant neoplasm among men in Western countries and the second leading cause of cancer mortalities after lung cancer. It is expected that by 2016, 180,890 men will be diagnosed with prostate cancer and 26,120 will die due to the disease. One of the most frequent complications of prostate cancer in patients is bone metastasis. Osteoblastic bone metastasis is the predominant finding at sites of skeletal metastases. Understanding the pathogenesis of prostate cancer progression and bone metastasis and targeting these processes are very important to control this aggressive disease. The main objectives of this work were to identify the role of signaling pathways that have shown to be upregulated in prostate cancer, evaluate a histone deacetylase inhibitor in treatment of prostate cancer metastasis and develop an osteoblastic PCa model for further understanding the molecular mechanisms of bone metastases. Intensive studies have been performed for better understanding the role of various signaling pathways in PCa progression and metastasis. Importantly, gastrin-releasing peptide receptor (GRPr) signaling was shown to be upregulated in human prostatic intraepithelial neoplasia (PIN), invasive prostatic carcinoma and related skeletal metastases. Our objective in this study was to determine the importance of this signaling in the pathogenesis of PCa progression. By activation of GRPr signaling using bombesin (BBN), there was an increase in proliferation and migration of canine prostate cancer cells. GRPR signaling also modulated the expression level of genes that are implicated in epithelial-mesenchymal transition (EMT), growth, invasion and metastasis of PCa. Histone deacetylase inhibitors (HDACi) are a class of therapeutics that can prevent tumor growth and induce differentiation and apoptosis in a wide group of cancers. AR-42, a HDACi, was shown to be efficient against multiple myeloma, lung cancer and hepatocellular carcino (open full item for complete abstract)

Committee: Thomas Rosol (Advisor); James DeWille (Committee Member); Beth Lee (Committee Member); Ahmad Shabsigh (Committee Member) Subjects: Animal Diseases; Animal Sciences; Biology; Medicine; Molecular Biology; Oncology; Pathology; Toxicology; Veterinary Services

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

Doctor of Philosophy, University of Toledo, 2014, Engineering

Bio-nanocomposites have recently attracted much interest in the field of bioengineering due to their exceptional chemical, mechanical and biological properties. On one hand, biopolymers such as chitosan have low mechanical properties which have to be improved by diverse methods in order to utilize them for medical applications. On the other hand, biocompatibility of the final material is critical for tissue engineering. Thus, fabricating and designing bio-nanocomposites with biocompatibility, biodegradability and improved mechanical properties are critical for next generation of implantable bone tissue biomaterials. Measurement of cell mechanical properties is also a timely and important topic in mechanical and bioengineering viewpoints. Previous studies have indicated there are some differences between healthy and cancerous cells which can be led to diagnosis of cancers. One of the main purposes of this dissertation is to improve the mechanical properties of chitosan using different methods of fabrication such as formation of cross-linking and addition of nanoparticles and tubes as reinforcement into chitosan matrix. In this study, Tripolyphosphate was used to cross-link between amino group in chitosan and phosphate groups of tripolyphosphate molecules. Micro- and nano- mechanical measurements showed that cross-linking chitosan would improve elastic modulus in both macro and nano scales. Atomic force microscopy based nanoindentation indicated that cross-linking decreased ductility of samples. In addition, surface morphology and material behavior under the applied loads were explored. A new method of measuring cell mechanical properties was introduced and the elastic modulus of two different cell lines at different cell regions was estimated using Hertz model. This novel method did not require any specific substrate treatment or cell fixation. The elastic modulus was measured at cell nucleus and cytoskeleton parts in human amniotic fluid stem cells and murine ost (open full item for complete abstract)

Committee: A. Champa Jayasuriya (Advisor); Ahalapitiya H. Jayatissa (Committee Co-Chair); Sorin Cioc (Committee Member); Matthew Franchetti (Committee Member); Mehdi Pourazady (Committee Member) Subjects: Biomedical Engineering; Materials Science; Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

The establishment and maintenance of the osteoblastic phenotype requires a complex network of transcription factors as well as signaling pathways. Some bone tumors seem to arise from genetic lesions that block differentiation of normal bone-forming osteoblasts. Patients with Carney Complex (CNC) are at risk for the development of osteochondromyxomas, caused by an immature expansion of osteoblasts precursors. Similarly, patients with McCune-Albright syndrome (MAS) develop fibrous dysplasia, a histologically similar bone lesion. Interestingly, it is known that uncontrolled cAMP-dependent protein kinase (PKA) signaling is involved in both MAS and CNC. In addition to this genetic evidence, proper regulation of PKA signaling has been demonstrated functionally to be essential for bone mineralization both in vitro and in vivo, although different effects have been observed. The work presented in this dissertation is focused on the involvement of PKA signaling itself as well as its crosstalk with the Wnt pathway in osteoblast biology. PRKAR1A is the gene encoding the type 1A regulatory subunit of PKA and it is the cause of CNC. Data from our lab has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through failure of terminal differentiation. In chapter two we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. Further, because Runx2 is a key mediator of (open full item for complete abstract)

Committee: Lawrence Kirschner (Advisor); Denis Guttridge (Committee Member); Virginia Sanders (Committee Member); Qianben Wang (Committee Member) Subjects: Cellular Biology; Molecular Biology

Master of Science in Chemical Engineering, Cleveland State University, 2013, Fenn College of Engineering

Under this research, live cell imaging of osteoblast-like marrow stromal cells has been carried out on polished and nanotextured (NaOH-etched) medical-grade titanium alloy (Ti-6Al-4V) surfaces to examine cellular adhesion and migration. The purpose of this research was to: 1) Build and assemble suitable hardware and software to conduct live cell imaging in a micro-incubator over an extended period of time. 2) Monitor and record live osteoblast-like marrow stromal cells on polished and NaOH-etched titanium alloy surfaces from cell inoculation to about one week of culture. 3) Measure location, area and perimeter of individual cells as a function of time, and examine if, as compared / contrasted with the polished titanium surface, that the NaOH-etched titanium surface promotes adhesion and migration of cells. This was achieved by describing the mobility, morphology and overall behavior of osteoblast-like marrow stromal cells. During the cell growth cycles, data generated from image analysis included the cells' center of mass (X,Y), their area, perimeter and shape as a function of incubation time. From the change in center of mass after each 15-minute interval, the real time speed of the cells was obtained. Major observations to support comparison studies between the surfaces determined that compared with polished titanium, NaOH-etched titanium promotes cellular filopodia growth, thus, promotes attachment. Filopodia provide cellular anchoring support and when prevalent, make cells more angular in shape. The median aspect ratio (length / width) of cells was found to be 1.38 on polished and 2.36 on NaOH-etched titanium. This, in addition to lower mean circularity shape factor values of 0.26 ± 0.03 on polished and 0.11 ± 0.01 on NaOH-etched titanium imply that the nanotextured surface promotes growth of cells more anchored to the substrate. This is also confirmed by increased perimeters of cells found on the NaOH-etched surface (950.92 ± 84.88 μm) compared with perime (open full item for complete abstract)

Committee: Surendra Tewari PhD (Committee Chair); Joanne Belovich PhD (Committee Member); Ronald Midura PhD (Committee Member) Subjects: Biomedical Engineering

Master of Science, Miami University, 2013, Chemical, Paper and Biomedical Engineering

Bone tissue engineering is an emerging field that seeks to improve the treatment of bone defects by restoring the functions of bone using the body's natural healing processes. Polymer scaffolds seeded with osteoblast and growth factors is one technique that has shown the potential to speed the healing process and decrease the rehabilitation time from bone defects. The goal of this study is to create viable polymer/ceramic scaffolds through melt processing of polycaprolactone and hydroxyapatite and using polyethylene oxide as porogen. The results of this study show that melt processing of these materials is an effective method for creating stable scaffolds. The properties of these scaffolds can be altered by changing several factors including polymer ratio, ceramic and salt content, and the pressure applied during the fabrication process. Biological analysis shows that the scaffolds seeded with MC3T3-E1 cells are capable of facilitating cell attachment and proliferation in vitro over time.

Committee: Azizeh Yousefi PhD (Advisor); Paul James PhD (Advisor); Paul Urayama PhD (Committee Member); Jason Berberich PhD (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Polymers

PhD, University of Cincinnati, 2002, Medicine : Pathobiology and Molecular Medicine

Insulin-like growth factor I (IGF-I) exerts anabolic effects on bone and is thought to amplify other osteogenic signals. However, the cellular and molecular mechanisms that mediate the anabolic actions of IGF-I have been difficult to address experimentally primarily due to the complexity of the IGF system. To study the local actions of IGF-I and its regulation by binding protein 4 (IGFBP-4) in skeletal tissue in a physiological context, I have developed mouse models with either overexpression of IGFBP-4 in osteoblasts or conditional disruption of the gene encoding the type I IGF receptor, Igf1r gene, in osteoblasts. Mice with overexpression of IGFBP-4 in bone osteoblasts exhibited reduced bone formation and turnover and severely impaired skeletal growth with global growth retardation. These effects were attributed to the sequestration of IGF-I by IGFBP-4 with consequent impairment of IGF-I actions. Osteoblast-specific disruption of the Igf1r gene reduced trabecular bone volume and severely impaired mineralization of bone matrix. Deficient IGF-I signaling led to a compensatory state of accelerated trabecular bone turnover. These studies established valuable models to further investigate the role of IGF-I signaling in bone.

Committee: Dr. Thomas Clemens (Advisor) Subjects: Biology, Molecular

Master of Science in Bioengineering, University of Toledo, 2005, Bioengineering

Studies have shown that the mechanical properties of bone, as a composite material, depnd on the mineralization, crystallinity, molecular structure, and arrangement of the mineral crystals with the collagen matrix. [1,2] Knowing that the strength of any ocmposite material is intimately affected by the size and shape of reinforcing inclusions, it can be proposed thar the material level mechanical function of bone tissue can be altered by modifying the size and/or shape of carbonated hydroxyapatite crystals. [3,4] Polyelectrolytes (negatively and positively charged macromolecules) have been shown to alter the nucleation, growth, and the resulting morphology of mineral crystals in solution by limiting growth on specific crystal faces. [5-8] Some of these polyelectrolytes are in peptide form and possess biocompatible properties. Therefore, polyelectrolytes carry the potential of being administered in vivo with the intention of modulating bone's mechanical function by way of tailoring crystal geometry and size. This study assessed biocompatibility of polyelectrolytic agents on osteoblast-like cells as well as the capability of polyelectrolytic agent to alter crystal properties in bone nodules formed in vitro. It was hypothesized that collagen production would not be altered, but the viability and genetic expression will differ with polyelectrolytic treatment. Furthermore, it is hypothesized that the size and shape of crystals will also differ with polyelectrolyte treatment.

Committee: Ozan Akkus (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, 2010, Oral Biology

A model of the initial complement mediated inflammatory response to tissue engineering (TE) scaffolds is developed in this study. TE has been given increasing attention in cases of large defects caused by severe trauma or disease based on the concept that the scaffold degrades and is replaced by tissue regeneration. Numerous attempts have been made to modify biomaterials chemical compositions, mechanical properties, and biomolecular incorporation to improve biocompatibility. The surface properties have been shown to affect host inflammatory responses and the ultimate fibrous capsule formation through foreign body reaction around implanted biomaterials. Although extensive research has been conducted regarding immune responses, such as macrophage activity, upon biomaterial particles and to a lesser extent on biomaterials surface, the inflammatory cascade and its effect on differentiation of preosteoblasts is not completely understood. Biomaterials are known to adsorb serum proteins and activate the complement system followed by subsequent inflammatory cascade. Complement activation through either direct deposition or interaction with adsorbed protein layer on biomaterial surface is believed to be the initiation process of inflammatory responses to implanted biomaterials. By setting the goal of constructing a tissue engineering model, the present study utilizes complement, macrophages, and preosteoblast-like cells to investigate the initial step of the inflammatory response and their effect on osteogenesis on biomaterial surface, poly(methylmethacrylate). PMMA polymer with different tacticities effects on MC3T3-E1 cell proliferation and differentiation were evaluated with cell proliferation and viability assay (MTS assay). In addition, their effect on MC3T3-E1 cell differentiation was assessed with MTS assay and osteogenic activity markers quantifications. No significant differences between different tacticity PMMA and tissue culture polystyrene were found. The effect (open full item for complete abstract)

Committee: Scott Schricker (Committee Chair); William Brantley (Committee Member); Sudha Agarwal (Committee Member); Binnaz Leblebicioglu (Committee Member) Subjects: Materials Science

Master of Science in Chemical Engineering, Cleveland State University, 2008, Fenn College of Engineering

Adhesion and proliferation of UMR 106-01 osteoblast cells were studied on various surface modified titanium materials such as polished, sandblasted, anodized and alkaline treated. Anodization of polished surface in Hydrofluoric acid developed nano-tubes, while NaOH treatment produced spongy microporous morphology. Test samples were coated with non-adhesive protein bovine serum albumin and compared with fibronectin coated specimens. The adhesion study lasted for 4 hrs, where osteoblast cells were cultured in serum free medium. Polished titanium, anodized titanium and NaOH titanium have shown similar percentages of cell adherence. The proliferation study lasted for 48 hrs, where cells were initially allowed to adhere to the surface in serum free medium for 4 hrs, followed by a medium change to 10% fatal bovine serum. The specific growth rate after 48 hrs in culture on the polished surface was found to be comparable to the tissue culture plastic, which exhibited a high growth rate. No significant difference was found in cell numbers between polished, anodized and NaOH-Ti, but each has varying cell orientation on the surface. Fluorescence images stained with alkaline phosphatase revealed that polished surface had cells flattened to the surface with short filapodia. Anodized surface had cells uniformly distributed across the surface where as NaOH-Ti displayed cells in colonies. Cells were found bonding to the surface of NaOH-Ti firmly using their filapodia as an anchoring agent. These results suggest that NaOH-Ti provides support in initial hours of implantation and bolsters cell proliferation. All together this process may help to better integrate titanium implant surfaces.

Committee: Joanne Belovich (Committee Chair); Ronald Midura (Committee Member); Surendra Tewari (Committee Member) Subjects: Biomedical Research; Chemical Engineering; Dental Care; Materials Science

Master of Sciences, Case Western Reserve University, 2008, Physiology and Biophysics

The Unfolded Protein Response (UPR) within the Endoplasmic Reticulum (ER) is a quality control mechanism ensuring properly folded proteins. OASIS and XBP-1 are two signal carriers of the UPR. The UPR is linked to tissue and cancer development. Elevated OASIS and XBP-1 activity are observed within developing osteoblasts. Additionally, heightened OASIS activity is present within osteosarcoma while sustained XBP-1 activity, unreported in osteosarcoma, is observed in other cancers. These signaling proteins may mediate development of mature osteoblasts and osteosarcomas. Therefore, evidence is presented with supporting mechanistic hypotheses indicating unique functions for OASIS and XBP-1 in osteoblast differentiation and osteosarcoma. These functions include, but are not limited to, enlarging the ER, buffering unfolded protein accumulation, mitigating UPR-induced cell death, and processing soluble ER proteins destined for secretion.

Committee: Edward Greenfield (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2006, Genetics

Low-density-lipoprotein receptor related protein 5 (LRP5) is a single pass transmembrane receptor that belongs to LDL receptor super-family. One function of LRP5 is to bind to a family of secreted glycoproteins (Wnt ligands) and transduce Wnt signaling. Mutations in LRP5 cause the autosomal recessive Osteoporosis-Pseudoglioma syndrome (OPPG), which is characterized by skeletal fragility due to markedly reduced bone mineral density and by congenital or childhood-onset of blindness. Additional mutations in LRP5 result in autosomal dominant high bone mass (HBM) diseases that are characterized by increased bone mineral density and a reduced incidence of skeletal fracture. This work describes in vitro biochemical studies that delineate the mechanism by which mutations cause HBM (chapter 2), the clinical and molecular features of OPPG (chapter 3), and in vivo studies of Lrp5 knock-out mice to understand the roles of the receptor in osteoblast function and their response to mechanical stress (chapter 4). Since LRP5 protein plays an important role in maintaining healthy bone mineral content, the findings presented in this thesis advanced our understanding of the mechanisms of several rare skeletal diseases, and provided new insights into common skeletal disorders, such as osteoporosis.

Committee: Matthew Warman (Advisor) Subjects: Biology, Genetics

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2013, College of Medicine

Smoking is a well established factor in many diseases and has multiple systemic effects due to more than 4000 different molecular species present in cigarette smoke. Osteoporosis, low bone mineral density, increased nonunion and delayed union, and increased risk of bone fracture have been associated with cigarette smoking. Nicotine, the main component of cigarette smoke is responsible for addiction. Previous studies have demonstrated deleterious impact of nicotine on skeletal remodeling and bone metabolism. Although many studies in the past have used osteoblasts and osteoblast like cells to study the impact of nicotine on bone, the use of human mesenchymal stem cells to determine the effects of nicotine has been rare. Therefore, learning whether or not these cells that go on to differentiate into osteoblasts and chondrocytes are also affected by nicotine will be particularly valuable in predicting the prognosis of a smoker undergoing orthopedic surgery/procedure. In this context, we examined the impact of nicotine in physiological range (0.1µM to 10 µM) on: a) hMSC proliferation b) Calcium deposition by osteoblasts (Alizarin red staining) c) Alkaline phosphatase activity (ALP assay on day) d) expression of canonical genes during differentiation of hMSCs (western blot analysis). Our results demonstrated a dose dependent decrease in hMSC proliferation, calcium deposition, ALP activity and expression of BMP-2 and HO-1. Interestingly, induction of heme oxygenase-1 (HO-1) by peroxisome proliferator-activated receptor delta (PPARd) agonist, GW0742, prevented the negative effect of nicotine. These results led to the conclusion that nicotine has a damaging effect on hMSCs proliferation and osteogenic differentiation and the induction of HO-1 by GW0742 results in the reversal of these effects. This offers an opportunity for HO-1 inducers to be used as therapeutic agents to improve bone fusion and fracture healing in smokers and non-smokers.

Committee: Nabil Ebraheim MD (Committee Chair); Jiayong Liu MD (Committee Member); Nitin Puri MD, PhD (Committee Member) Subjects: Biomechanics; Biomedical Research; Cellular Biology