Skip to Main Content
 

Global Search Box

 
 
 
 

ETD Abstract Container

Abstract Header

The Effects of Recombinant Osteoactivin on Murine Osteoclastogenesis

Khol, Matthew Philip

Abstract Details

2014, Master of Science, Northeast Ohio Medical University, Integrated Pharmaceutical Medicine.
ABSTRACT Introduction: Healthy bone constantly remodels, in balance between osteoclast bone resorption and osteoblast bone formation. Osteoclasts differentiate in vitro from osteoclast progenitor cells in the presence of macrophage-colony stimulating factor and receptor activated nuclear factor κB-ligand, while osteoblasts differentiate in vitro from mesenchymal stem cells in the presence of ascorbic acid, β-glycerol phosphate, and dexamethasone. Imbalance between the function of osteoclasts and osteoblasts results in disease. In osteoporosis, bone resorption dominates, leading to decreased bone mass. Osteoporosis is of critical concern to an aging population, and new treatments are needed. Osteoactivin is an anabolic bone growth factor in vivo, causing increased bone mass when injected into mice. In vitro, osteoactivin has been shown to increase osteoblast differentiation and function. However, the effects of osteoactivin on osteoclasts are not as well defined. In this study, we hypothesized that recombinant osteoactivin inhibits osteoclastogenesis. In the future, osteoactivin could be a therapeutic agent for bone healing and preventing or even reversing osteoporosis. Material and Methods: Osteoclast progenitor cells were isolated from C57BL/6 murine tibias, femurs, and humeri. Several aspects of osteoclastogenesis were tested in the presence of dose-dependent recombinant osteoactivin. First, proliferation and survival assays, with and without macrophage-colony stimulating factor respectively, were conducted on the osteoclast progenitor cells in the presence of recombinant osteoactivin. Next, the osteoclast progenitor cells were differentiated into osteoclasts in the presence of recombinant osteoactivin. Osteoclast tartrate resistant acid phosphatase activity was measured and stained as a marker for terminally differentiated osteoclasts. Differential cell counting was performed for osteoclasts containing 3-20 and >20 nuclei. Two functional assays, Corning surface and cortical bovine bone slices, were performed as well in order to quantify osteoclast resorption activity in the presence of recombinant osteoactivin. Finally, osteoclast survival in the presence of recombinant osteoactivin was tested without receptor activated nuclear factor κB-ligand. Results: Osteoclast progenitor cell proliferation and survival showed no differences from control when treated with recombinant osteoactivin. Likewise, when recombinant osteoactivin was added on the first day of osteoclast differentiation, there were negligible changes. However, when administered on day 4 or day 6 of differentiation, recombinant osteoactivin treatment resulted in fewer osteoclasts with >20 nuclei. In the Corning functional assay, recombinant osteoactivin administered on day 1 and day 4 of differentiation increased resorption, but day 6 administration decreased resorption, although not significantly. Meanwhile, in the cortical bone slice functional assay, pre-formed osteoclasts exhibited decreased resorption with recombinant osteoactivin, although not significantly. Finally, the osteoclast survival assay with recombinant osteoactivin showed little effect at 24hrs, but an increased number of osteoclasts with >20 nuclei after 48hrs, although the results were again not significant. Conclusions: These data support recombinant osteoactivin as an inhibitor of osteoclast differentiation and function, although the functional data were inconclusive. Osteoclast differentiation was clearly inhibited by recombinant osteoactivin. Further, differentiation data suggested that recombinant osteoactivin is an inhibitor of osteoclast cell-cell fusion, as evidenced by fewer terminally differentiated osteoclasts with >20 nuclei. Osteoclast functional tests were inconclusive. Recombinant osteoactivin increased osteoclast resorption of Corning surface when added on day 1 and day 4 of differentiation. However, this may be due to endogenous osteoactivin, as recombinant osteoactivin has been washed away before osteoclasts were formed. Endogenous osteoactivin appears to have been down-regulated and/or its receptors occupied due to earlier high levels of recombinant osteoactivin. Supporting this conclusion, resorption was inhibited by administration of recombinant osteoactivin on day 6 of differentiation when osteoclasts were formed, although the data was not significant. Recombinant osteoactivin also decreased pre-formed osteoclast resorption of bovine bone slices, although not significantly. Recent data from our lab suggest osteoactivin decreases DC-STAMP expression in osteoclasts, a transmembrane protein required for osteoclast cell-cell fusion and bone resorption activity; it is possible that this protein is involved and responsible for the observed results. The osteoclast survival assay in the presence of recombinant osteoactivin suggested increased survival of large osteoclasts with >20 nuclei, although the results were not significant. Osteoclasts typically undergo apoptosis in about 3 days because their activity is so high. Perhaps, inhibition of osteoclast activity through DC-STAMP, also provides a protective effect from apoptosis. The initial hypothesis still stands: osteoclastogenesis is inhibited by recombinant osteoactivin. However, the osteoclast functional experiments must be repeated in order to achieve significance. Ultimately, this basic research could translate into novel treatments for the relatively common human ailment, osteoporosis, and improve health and quality of life for many.
Fayez Safadi, Ph.D. (Advisor)
Werner Geldenhuys, Ph.D. (Committee Member)
Moses Oyewumi, Ph.D. (Committee Member)
78 p.

Recommended Citations

Citations

  • Khol, M. P. (2014). The Effects of Recombinant Osteoactivin on Murine Osteoclastogenesis [Master's thesis, Northeast Ohio Medical University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ne2mh1424053380

    APA Style (7th edition)

  • Khol, Matthew. The Effects of Recombinant Osteoactivin on Murine Osteoclastogenesis. 2014. Northeast Ohio Medical University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ne2mh1424053380.

    MLA Style (8th edition)

  • Khol, Matthew. "The Effects of Recombinant Osteoactivin on Murine Osteoclastogenesis." Master's thesis, Northeast Ohio Medical University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ne2mh1424053380

    Chicago Manual of Style (17th edition)