Master of Science (M.S.) in Dentistry
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College of Dental Medicine
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Nova Southeastern University
Dana Walid Al Hashimi. 2018. Osteoblast and Osteoclast Bioactivity Responses to Growth Factors and Inhibitors. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, College of Dental Medicine. (120)
Bone healing, regeneration, maintenance, and bone defect repair is dependent on the regenerative capacity of Osteoblasts. Bone resorption, osteoporosis, peri implantitis and bone loss are caused by the activity of Osteoclasts. It is the balance in activity between bone regeneration by Osteoblasts and bone resorption by Osteoclasts which determines if bone regeneration or bone loss occurs. Osteoblast and Osteoclast cells respond to growth factors and bioactive inhibitors within the local environment, and the concentrations of these molecules may help determine if bone loss or bone regeneration occurs. To develop new therapies which can promote bone regeneration and avoid bone resorption: I studied the effects of three growth factors: Spingosine-1-Phosphate (S1P), Bone Morphogenic Protein-2 (BMP-2), and Growth Factor Beta 1 (TGF-B1) and several bioactive inhibitors: FTY720, K02288, A83-01, M5939, Osteostatin, Sclerostin, Odanacatic, Carfilzomib, Phloretin, Decitabine, Amiloride, Osteoprotegerin, Tacrolimus, Cabozantinib, Cathepsin inhibitor, GM 6001 and Osteostatin. To overcome the problem of the short lifespan of Osteoblasts and Osteoclasts: I used a lenti-virus to immortalize the cells to increase their lifespan, and I created two novel cell lines for my research, which I have named “Immortalized Osteoblasts” and “Immortalized Osteoclasts.” My research design comprised of 54 treatment groups and a total of 896 specimens. Several interesting novel results were observed, as follows: First, I investigated and compared the migration responses of immortalized Osteoblast and Osteoclast cells in response to three growth factors and four bioactive inhibitors using a transwell cell migration assay. I observed that S1P was the most effective promoter of Osteoblast and Osteoclast migration, because S1P was able to increase cell migration by almost 3,000% and 1,000% respectively. The most effective inhibitor of Osteoblast (94%) and Osteoclast (100%) migration was Sclerostin. Second, I investigated and compared the proliferation of immortalized Osteoblast and Osteoclast cells using a MTT spectrophotometer assay in response to growth factors and bioactive inhibitors. I observed that Phloretin was the most effective promoter of Osteoblast proliferation (394%), and that Amiloride was the most effective promoter of Osteoclast proliferation (899%). The most effective inhibitor of Osteoblast proliferation was GM6001 (-69%), and the most effective inhibitor of Osteoclast proliferation was Tacrolimus (-71%). Third, I investigated and compared the mineralization of bone by Osteoblasts using a spectrophotometer mineralization assay in response to growth factors and bioactive inhibitors. I observed that S1P was the most effective promoter of Osteoblast mineralization (408%). Fourth, I investigated and compared the degradation of Collagen by Osteoclasts using a Mouse Tail Collagen Type-1 spectrophotometer degradation assay in response to growth factors and bioactive inhibitors. I observed that FTY720 was the most effective compound at preventing collagen degradation (506%). Fifth, I compared the mean overall bioactive effects of many bioactive molecules on Osteoblasts and Osteoclasts, and discovered that the most bioactive was S1P. The average effect of S1P in six assays was 734%. The least effective compound was GM6001. The mean effect of GM6001 in six assays was -19%. Sixth, I designed a hypothetical scaffold comprising of S1P, Phloretin, and FTY720, that could be developed to be implanted into bone defects and empty tooth sockets to promote bone regeneration, and to prevent osteonecrosis, osteoporosis, and peri implantitis. Due to a lack of time and money, I was unable to investigate the effectiveness of my hypothetical scaffold, but it is my recommendation for future research in clinical trials to determine if it will benefit patients. My sincere hope is that patients, the dental profession, and manufacturers of osteoregenerative materials, scaffolds and membranes will benefit from the ideas developed in my thesis. I recognize that the treatment of bone defects, fractures, and infected bone can be difficult because of the long time it takes (months and years) to regenerate new replacement bone. Clearly a new generation of bioactive bone repair materials, scaffolds and membranes could have the potential to increase the speed and effectiveness of osteoregeneration, and to help inhibit osteoporosis, osteonecrosis, and peri implantitis.
Bone, Bone regeneration, Growth factors, Inhibitors, Osteoporosis, Peri implantitis
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