Effectiveness Of Single And Dual Release Of Melatonin And Bmp-2 In Bone Homeostasis

Abstract

In the present thesis, it was aimed to develop single and dual release systems to achieve the prolonged delivery of melatonin (MEL) and bone morphogenetic protein two (BMP-2) and to investigate their synergistic effect on bone homeostasis. Two different release systems were constructed. The first construct which consisted of chitosan and hydroxyapatite composite scaffolds (chitosan/HAp) embedded with MEL-loaded and BMP-2-loaded microparticles, was used to compare the efficacy of single and dual release on osteoblast differentiation. Meanwhile, the second construct which consisted of MEL and BMP-2 embedded chitosan/HAp scaffolds was used to investigate whether the osteoclast activation mediated by BMP-2 high dose could be minimized by the inhibitory effect of melatonin. For osteoblastogenesis studies, MEL and BMP-2 were first encapsulated in poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) by double emulsion solvent evaporation method using different formulations. After the characterization of MPs prepared by different formulations, MEL MPs and BMP-2 MPs which had diameters of approximately 2 µm and 11µm, respectively, were selected for cell culture studies. The in vitro cell culture studies were performed by seeding MC3T3-E1 cells into MEL MPs and/or BMP-2 MPs embedded chitosan/HAp scaffolds. Relative expression of alkaline phosphatase (ALP) and Runt Associated Transcription Factor 2 (RUNX2), mineralization detected by alizarin red and von Kossa stains were higher in scaffolds containing dual release system than that of single release ones. Additionally, according to haematoxylin and eosin staining and SEM analysis, number of cells were higher and morphology of cells was more similar to osteoblasts in dual release group than that of single release ones. Results suggest that the dual release of MEL and BMP-2 could enhance bone formation mediated by osteoblasts in more effective manner than when one of them was used. On the other hand, MEL and/or BMP-2 were directly embedded within chitosan/HAp scaffolds and their synergistic effect on osteoclastogenesis was investigated by seeding RAW 264.7 cells into embedded scaffolds. The higher number of tartrate resistant acid phosphatase positive (TRAP+) cells, the high expression of cathepsin K and nuclear factor of activated T cells one (NFATC1) genes, TRAP activity and the excessive formation of large osteoclasts in the fields of SEM images demonstrated that BMP-2-embedded scaffolds have enhanced osteoclast formation than that of MEL-embedded and dual release groups. However, relative expression of cathepsin K, NFATC1 and TRAP genes, TRAP activity, number TRAP+ cells and osteoclasts observed in TRAP staining and SEM, respectively, were lower in MEL and BMP-2 embedded scaffolds than that of BMP-2-embedded scaffolds. Results indicate that the inhibitory effect of melatonin on osteoclast formation has effectively minimized osteoclast formation caused by BMP-2 high dose. Taking all things into account, controlled release of MEL and BMP-2 from PLGA microparticles using relatively low doses has effectively enhanced bone formation by the activation of osteoblast formation. However, in some cases it would be more preferable when the engineered construct has both osteoblast-inductive and osteoclast-inhibitive properties and this is what has been demonstrated in the second system as melatonin has inhibited osteoclast formation induced by BMP-2 at the same time the used dose of BMP-2 could also induce osteoblast formation.