Kemik Doku Mühendisliği Yaklaşımında Melatoninin Rolü: İn Vitro Çalışmalar

Abstract

In the presented study, it was aimed to develop a tissue scaffold-based system in which the osteoinductive and anticarcinogenic properties of melatonin coexist. For this purpose, osteoinductive effects of melatonin were investigated using preosteoblatic MC3T3-E1 cell line and human mesenchymal stem cells (hMSC); anticarcinogenic effects of melatonin were investigated using MG-63 human osteosarcoma cell line by in vitro cell culture studies. It has been determined that, poly(lactide-co-glycolic acid) (PLGA) nanoparticles and PLGA microparticles produced by emulsion/solvent evaporation method have diameters of about 200 nm and 3 μm, respectively. In vitro release studies showed that, PLGA nano/microparticles are highly suitable systems for controlled and long-term release of melatonin, and the concentration of melatonin released from the particles after 40 days is in micromolar level. The release behavior of melatonin from the particles was consistent with the Higuchi release model, so the diffusional mechanism proved to be the result of mathematical analysis. PLGA nanoparticles were shown to be toxic on both preosteoblastic MC3T3-E1 cells and MG-63 human osteosarcoma cells and the percentages of particles uptaken by the cells were determined to be ~ 30% and ~ 60%, respectively. Therefore, it has been emphasized that PLGA nanoparticles should be loaded onto tissue scaffolds. In order to increase the bioactivity of chitosan, chitosan/hydroxyapatite (HAp) tissue scaffolds were produced by freeze-drying by adding HAp particles in powder and bead form to the chitosan structure. Three-dimensional pore structures of the scaffolds were investigated by scanning electron microscopy (SEM) and micro-computerized tomography (μ-CT). Studies conducted with MC3T3-E1 cells have shown that chitosan/bead HAp tissue scaffold more strongly supported cell proliferation. It was determined that, cells proliferating in chitosan/HAp+Mp tissue scaffolds prepared by incorporation of melatonin loaded PLGA microparticles during the production of chitosan/HAp tissue scaffolds produced the most favorable results. The alkalene phosphatase (ALP) activity and the expression levels of runt-related transcription factor (RunX2), collagen type 1 (Col1), osteocalcin (Ocn) and osteopontin (Opn) genes of this group were found to be higher than that of the other groups. SEM and confocal laser scanning microscope (CLSM) images showed that the cells migrated into the depth of 600-700 μM of the scaffold. To investigate the effect of melatonin on osteosarcoma cells, melatonin/hydroxypropyl-β-cyclodextrin (HPβCD) inclusion complexes were produced under 900 W, 60 s and 90 s microwave conditions. Melatonin/HPβCD inclusion complexes were successfully loaded into chitosan/HAp+Mp to contain 2.267 ± 0.236 mg of melatonin per scaffold. In vitro release studies demonstrated that melatonin was released from tissue scaffolds within a very short time period such as 5 days and the released melatonin concentration was approximately 8 mM. The effect of melatonin/HPβCD inclusion complex-loaded tissue scaffolds on MG-63 human osteosarcoma cells was examined by in vitro cell culture studies and it was determined that melatonin released from this system in a high amount and very rapidly inhibited the proliferation of cancer cells in the G0/G1 phase of the cell cycle. Silk films were used to study the effect of melatonin on the differentiation of hMSCs into osteoblasts and the surface of these films was modified with melatonin at different concentrations (0-2,000 μM). In the first 24 hours, most of the melatonin was released suddenly and the release continued for 5 days. Since concentrations of melatonin at 250 μM, 500 μM and 1,000 μM did not cause any toxic effects on the cells, in vitro cell culture studies with hMSCs were carried out using these concentrations. Studies of both growth and differentiation medium showed that, the presence of melatonin increased ALP activities of cells and RUNX2 and OCN protein expressions that were early and late differentiation markers, respectively. As a result of Von Kossa staining, it was determined that cells cultured on silk films containing melatonin synthesized mineralized nodules in the late culture period. As a result of the obtained data, it was determined that melatonin in the micromolar concentration released from the developed tissue scaffold-based melatonin carrier system increased the proliferation of preosteoblast cells and melatonin in milimolar concentration inhibited the proliferation of osteosarcoma cells. Thus, it was concluded that this system, in which osteoinductive and anticarcinogenic properties of melatonin are effective, is suitable for osteosarcoma treatment.