In vitroevaluation of osteoprotegerin in chitosan for potential bone defect applications

Abstract

BackgroundThe receptor activator of nuclear factor kappa-B (RANK)/RANK ligand/osteoprotegerin (OPG) system plays a critical role in bone remodelling by regulating osteoclast formation and activity. OPG has been used systemically in the treatment of bone diseases. In searching for more effective and safer treatment for bone diseases, we investigated newly formulated OPG-chitosan complexes, which is prepared as a local application for its osteogenic potential to remediate bone defects.MethodsWe examined high, medium and low molecular weights of chitosan combined with OPG. The cytotoxicity of OPG in chitosan and its proliferation in vitro was evaluated using normal, human periodontal ligament (NHPL) fibroblasts in 2D and 3D cell culture. The cytotoxicity of these combinations was compared by measuring cell survival with a tetrazolium salt reduction (MTT) assay and AlamarBlue assay. The cellular morphological changes were observed under an inverted microscope. A propidium iodide and acridine orange double-staining assay was used to evaluate the morphology and quantify the viable and nonviable cells. The expression level of osteopontin and osteocalcin protein in treated normal human osteoblast cells was evaluated by using Western blot.ResultsThe results demonstrated that OPG in combination with chitosan was non-toxic, and OPG combined with low molecular weight chitosan has the most significant effect on NHPL fibroblasts and stimulates proliferation of cells over the period of treatment.

Osteoprotegerin (OPG) pathways in bone diseases and its application in therapeutic perspectives

“…It was previously reported that OPG-chitosan matrices and gel increase the in vitro proliferation of normal human osteoblasts and fibroblasts cells that used later to treat the critical size defects on parietal bone of rabbit. The results of this studies showed that bioresorbable OPGchitosan material induced the formation of a significant quantity of bone in a critical-sized parietal bone defect in a rabbit model [72][73][74].…”

Evaluation of the glycemic effect ofCeratonia siliquapods (Carob) on a streptozotocin-nicotinamide induced diabetic rat model

“…After 24 h of incubation, decreasing concentrations of carob methanolic extract (such as 1,000, 500, 250, 125, 60, 30, 15 and 7 µmol) were prepared (0.025% DMSO) and transferred to the cells with incubating for 24, 48,72 h at 37 °C and 5% CO 2 . Subsequently, 20 µL of MTT solution (5 mg mL −1 ) was added to the treated cells in a dark place, covered with aluminum foil and incubated for 4 h. All media was discharged and a total of 100 µL of DMSO was poured into each well until the purple formazan crystals dissolved ( Jayash et al, 2016 ; Jayash et al, 2017 ). The blank was performed in a similar manner but the test samples were replaced by media (0.025% DMSO).…”

Effect of <i>Catha edulis</i> (khat) on pancreatic functions in streptozotocin-induced diabetes in male Sprague-Dawley rats

“…In vitro cytotoxicity of the ethanolic extract of khat Cell culture: RIN-14B (rat pancreatic islets cell tumor) and WRL 68 (human normal liver) cells were sub-cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich, St. Louis, MO, USA) and RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), respectively. The media were supplemented with 10% FBS and 1% penicillin-streptomycin [ 26 ].…”